Compounds αvβ6 integrin antagonists

ABSTRACT

A compound of formula (I): 
                         
being 4-(3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl) ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid, or a salt thereof.

FIELD OF THE INVENTION

The present invention relates to pyrrolidine compounds being α_(v)β₆integrin antagonists, pharmaceutical compositions comprising suchcompounds and to their use in therapy, especially in the treatment ofconditions for which an α_(v)β₆ integrin antagonist is indicated, forthe use of a compound in the manufacture of a medicament for thetreatment of conditions in which an antagonist of α_(v)β₆ integrin isindicated and a method for the treatment or prophylaxis of disorders inwhich antagonism of α_(v)β₆ integrin is indicated in a human.

BACKGROUND OF THE INVENTION

Integrin superfamily proteins are heterodimeric cell surface receptors,composed of an alpha and beta subunit. At least 18 alpha and 8 betasubunits have been reported, which have been demonstrated to form 24distinct alpha/beta heterodimers. Each chain comprises a largeextracellular domain (>640 amino acids for the beta subunit, >940 aminoacids for the alpha subunit), with a transmembrane spanning region ofaround 20 amino acids per chain, and generally a short cytoplasmic tailof 30-50 amino acids per chain. Different integrins have been shown toparticipate in a plethora of cellular biologies, including cell adhesionto the extracellular matrix, cell-cell interactions, and effects on cellmigration, proliferation, differentiation and survival (Barczyk et al,Cell and Tissue Research, 2010, 339, 269).

Integrin receptors interact with binding proteins via shortprotein-protein binding interfaces. The integrin family can be groupedinto sub-families that share similar binding recognition motifs in suchligands. A major subfamily is the RGD-integrins, which recognise ligandsthat contain an RGD (arginine-glycine-aspartic acid) motif within theirprotein sequence. There are 8 integrins in this subfamily, namelyα_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, α_(IIb)β₃, α₅β₁, α₈β₁,where nomenclature demonstrates that α_(v)β₁, α_(v)β₃, α_(v)β₅, α_(v)β₆,& α_(v)β₈ share a common av subunit with a divergent β subunit, andα_(v)β₁, α₅β₁ & α₈β₁ share a common β₁ subunit with a divergent asubunit. The β₁ subunit has been shown to pair with 11 different αsubunits, of which only the 3 listed above commonly recognise the RGDpeptide motif (Humphries et al, Journal of Cell Science, 2006, 119,3901).

The 8 RGD-binding integrins have different binding affinities andspecificities for different RGD-containing ligands. Ligands includeproteins such as fibronectin, vitronectin, osteopontin, and the latencyassociated peptides (LAPs) of Transforming Growth Factor β₁ and β₃(TGFβ₁ and TGFβ₃). Integrin binding to the LAPs of TGFβ₁ and TGFβ₃ hasbeen shown in several systems to enable activation of the TGFβ₁ andTGFβ₃ biological activities, and subsequent TGFβ-driven biologies(Worthington et al, Trends in Biochemical Sciences, 2011, 36, 47). Thediversity of such ligands, coupled with expression patterns ofRGD-binding integrins, generates multiple opportunities for diseaseintervention. Such diseases include fibrotic diseases (Margadant et al,EMBO reports, 2010, 11, 97), inflammatory disorders, cancer(Desgrosellier et al, Nature Reviews Cancer, 2010, 10, 9), restenosis,and other diseases with an angiogenic component (Weis et al, ColdSpring. Harb. Perspect. Med. 2011, 1, a 006478).

A significant number of α_(v) integrin antagonists (Goodman et al,Trends in Pharmacological Sciences, 2012, 33, 405) have been disclosedin the literature including inhibitory antibodies, peptides and smallmolecules. For antibodies these include the pan-α_(v) antagonistsIntetumumab and Abituzumab (Gras, Drugs of the Future, 2015, 40, 97),the selective α_(v)β₃ antagonist Etaracizumab, and the selective α_(v)β₆antagonist STX-100. Cilengitide is a cyclic peptide antagonist thatinhibits both α_(v)β₃ and α_(v)β₅ and SB-267268 is an example of acompound (Wilkinson-Berka et al, Invest. Ophthalmol. Vis. Sci., 2006,47, 1600), that inhibits both α_(v)β₃ and α_(v)β₅. Invention ofcompounds to act as antagonists of differing combinations of α_(v)integrins enables novel agents to be generated tailored for specificdisease indications.

Pulmonary fibrosis represents the end stage of several interstitial lungdiseases, including the idiopathic interstitial pneumonias, and ischaracterised by the excessive deposition of extracellular matrix withinthe pulmonary interstitium. Among the idiopathic interstitialpneumonias, idiopathic pulmonary fibrosis (IPF) represents the commonestand most fatal condition with a typical survival of 3 to 5 yearsfollowing diagnosis. Fibrosis in IPF is generally progressive,refractory to current pharmacological intervention and inexorably leadsto respiratory failure due to obliteration of functional alveolar units.IPF affects approximately 500,000 people in the USA and Europe.

There are in vitro experimental, animal and IPF patientimmunohistochemistry data to support a key role for the epitheliallyrestricted integrin, α_(v)β₆, in the activation of TGFβ1. Expression ofthis integrin is low in normal epithelial tissues and is significantlyup-regulated in injured and inflamed epithelia including the activatedepithelium in IPF. Targeting this integrin, therefore, reduces thetheoretical possibility of interfering with wider TGFβ homeostaticroles. Partial inhibition of the α_(v)β₆ integrin by antibody blockadehas been shown to prevent pulmonary fibrosis without exacerbatinginflammation (Horan G S et al Partial inhibition of integrin α_(v)β₆prevents pulmonary fibrosis without exacerbating inflammation. Am JRespir Crit Care Med 2008 177: 56-65). Outside of pulmonary fibrosis,α_(v)β₆ is also considered an important promoter of fibrotic disease ofother organs, including liver and kidney (Reviewed in Henderson N C etal Integrin-mediated regulation of TGFβ in Fibrosis, Biochimica etBiophysica Acta—Molecular Basis of Disease 2013 1832:891-896),suggesting that an α_(v)β₆ antagonist could be effective in treatingfibrotic diseases in multiple organs.

Consistent with the observation that several RGD-binding integrins canbind to, and activate, TGFβ, different α_(v) integrins have recentlybeen implicated in fibrotic disease (Henderson N C et al Targeting ofα_(v) integrin identifies a core molecular pathway that regulatesfibrosis in several organs Nature Medicine 2013 Vol 19, Number 12:1617-1627; Sarrazy V et al Integrins αvβ5 and αvβ3 promote latent TGF-β1activation by human cardiac fibroblast contraction Cardiovasc Res 2014102:407-417; Minagawa S et al Selective targeting of TGF-β activation totreat fibroinflammatory airway disease Sci Transl Med 2014 Vol 6, Issue241: 1-14; Reed N I et al. The α_(v)β₁ integrin plays a critical in vivorole in tissue fibrosis Sci Transl Med 2015 Vol 7, Issue 288: 1-8).Therefore inhibitors against specific members of the RGD bindingintegrin families, or with specific selectivity fingerprints within theRGD binding integrin family, may be effective in treating fibroticdiseases in multiple organs.

SAR relationships of a series of integrin antagonists against α_(v)β₃α_(v)β₅, α_(v)β₆ and α_(v)β₈ have been described (Macdonald, S J F etal. Structure activity relationships of α_(v) integrin antagonists forpulmonary fibrosis by variation in aryl substituents. ACS Med Chem Lett2014, 5, 1207-1212. 19 Sep. 2014).

It is an object of the invention to provide α_(v)β₆ inhibitors,preferably with activities against other α_(v) integrins, such asα_(v)β₁, α_(v)β₃, α_(v)β₅ or α_(v)β₈.

BRIEF SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a compoundof formula (I), 4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl) ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl) butanoic acid or a salt thereof, more particularly a compound offormula (I) or a pharmaceutically acceptable salt thereof:

Compounds of formula (I) and their salts have α_(v)β₆ antagonistactivity and are believed to be of potential use for the treatment orprophylaxis of certain disorders.

The term α_(v)β₆ antagonist activity includes α_(v)β₆ inhibitor activityherein.

In a second aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable carriers, diluents or excipients.

In a third aspect of the present invention, there is provided a compoundof formula (I), or a pharmaceutically acceptable salt thereof for use intherapy, in particular in the treatment of a disease or condition forwhich an α_(v)β₆ integrin receptor antagonist is indicated.

In a fourth aspect of the present invention, there is provided a methodof treatment or prophylaxis of a disease or condition for which anα_(v)β₆ integrin receptor antagonist is indicated in a human in needthereof which comprises administering to a human in need thereof atherapeutically effective amount of compound of formula (I) or apharmaceutically acceptable salt thereof.

In a fifth aspect of the present invention, there is provided the use ofa compound of formula (I), or a pharmaceutically acceptable salt thereofin the manufacture of a medicament for the treatment of a disease orcondition for which an α_(v)β₆ integrin receptor antagonist isindicated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the X-ray crystal structure of compound (XVIII).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect of the present invention, there is provided a compoundof formula (I),4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl) ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid, or a saltthereof, more particularly a compound of formula (I) or apharmaceutically acceptable salt thereof:

In another embodiment the compound of Formula (I) is a pharmaceuticallyacceptable salt of 4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid.

In another embodiment the compound of Formula (I) is4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid.

In an embodiment the compound of formula (I) has the formula (IA1):

-   being    (R)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)    ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid    or a pharmaceutically acceptable salt thereof.

In an embodiment the compound of formula (I) has the formula (IA2):

-   being    (S)-4-((S)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)    ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid    or a pharmaceutically acceptable salt thereof

In an embodiment the compound of formula (I) has the formula (IA3):

-   being    (R)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)    ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid    or a pharmaceutically acceptable salt thereof.

In an embodiment the compound of formula (I) has the formula (IA4):

-   being    (S)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)    ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid    or a pharmaceutically acceptable salt thereof.

In another embodiment the compound of Formula (I) or any one ofcompounds IA1, IA2, IA3 or IA4 is a pharmaceutically acceptable salt of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid.

Compounds of formula (I) have both a basic amine group and a carboxylicacid group and can consequently form an internal salt i.e. a zwitterionor inner salt. Therefore in an embodiment the compound of formula (I) is4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid or any one of compounds IA1, IA2, IA3 or IA4 in a zwitterion saltform. In another embodiment, the compound of formula (I) is4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid or any one of compounds IA1, IA2, IA3 or IA4 in a non-zwitterionicform.

It will be appreciated that the present invention covers compounds offormula (I), IA1, IA2, IA3 or IA4 as the parent compound, as azwitterion (the parent compound is protonated internally by itscarboxylic acid group and normally exists as a zwitterion) and as saltsthereof, for example as a pharmaceutically acceptable salt thereof.

For a review on suitable salts see Berge et al., J. Pharm. Sci.,66:1-19, (1977). Suitable pharmaceutically acceptable salts are listedin P H Stahl and C G Wermuth, editors, Handbook of Pharmaceutical Salts;Properties, Selection and Use, Weinheim/Zurich: Wiley-VCH/VHCA, 2002.Suitable pharmaceutically acceptable salts can include acid additionsalts with inorganic acids such, for example, as hydrochloric acid,hydrobromic acid, orthophosphoric acid, nitric acid, phosphoric acid, orsulphuric acid, or with organic acids such, for example asmethanesulphonic acid, ethanesulphonic acid, p-toluenesulphonic acid,acetic acid, propionic acid, lactic acid, citric acid, fumaric acid,malic acid, succinic acid, salicylic acid, maleic acid,glycerophosphoric acid, tartaric, benzoic, glutamic, aspartic,benzenesulphonic, naphthalenesulphonic such as 2-naphthalenesulphonic,hexanoic acid or acetylsalicylic acid, in particular, maleic acid.Typically, a pharmaceutically acceptable salt may readily be prepared byusing a desired acid or base as appropriate. The resultant salt mayprecipitate from solution and be collected by filtration or may berecovered by evaporation of the solvent.

Other non-pharmaceutically acceptable salts, e.g. formates, oxalates ortrifluoroacetates, may be used, for example in the isolation of thecompounds of formula (I), and are included within the scope of thisinvention.

A pharmaceutically acceptable base addition salt can be formed byreaction of a compound of formula (I) with a suitable organic base,(e.g. triethylamine, ethanolamine, triethanolamine, choline, arginine,lysine or histidine), optionally in a suitable solvent, to give the baseaddition salt which is usually isolated, for example, by crystallisationand filtration. Pharmaceutically acceptable inorganic base salts includeammonium salts, alkali metal salts such as those of sodium andpotassium, alkaline earth metal salts such as those of calcium andmagnesium and salts with organic bases, including salts of primary,secondary and tertiary amines, such as isopropylamine, diethylamine,ethanolamine, trimethylamine, dicyclohexyl amine andN-methyl-D-glucamine.

In one embodiment the compound of formula (I) is in the form of theparent compound, for example4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl) ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compounds of formula (I).

The compounds of formula (I) may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of formula(I) may exist as polymorphs, which are included within the scope of thepresent invention. Polymorphic forms of compounds of formula (I) may becharacterized and differentiated using a number of conventionalanalytical techniques, including, but not limited to, X-ray powderdiffraction (XRPD) patterns, infrared (IR) spectra, Raman spectra,differential scanning calorimetry (DSC), thermogravimetric analysis(TGA) and solid state nuclear magnetic resonance (SSNMR).

The compounds of formula (I) may also be prepared as an amorphousmolecular dispersion in a polymer matrix, such as hydroxypropylmethylcellulose acetate succinate, using a spray-dried dispersion (SDD)process to improve the stability and solubility of the drug substance.

The compounds of formula (I) may also be delivered using a liquidencapsulation technology to improve properties such as bioavailabilityand stability, in either liquid or semi-solid filled hard capsule orsoft gelatin capsule formats.

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallized. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or capable of forming hydrogen bonds such aswater, xylene, N-methyl pyrrolidinone, methanol and ethanol may be usedto form solvates. Methods for identification of solvates include, butare not limited to, NMR and microanalysis. It will be appreciated thatcrystalline forms optionally may be solvated to form, for example,pharmaceutically acceptable solvates, such as hydrates which may bestoichiometric hydrates as well as compounds containing variable amountsof water. Solvates include stoichiometric solvates andnon-stoichiometric solvates. Compounds of formula (I) may exist insolvated or non-solvated form.

The compounds described herein contain two asymmetric centres so thatoptical isomers, e.g. diastereoisomers and enantiomers may be formed.Accordingly, the present invention encompasses isomers of the compoundsof formula (I) whether as individual isomers isolated such as to besubstantially free of the other isomer (i.e. pure) or as mixtures. Anindividual isomer isolated such as to be substantially free of the otherisomer (i.e. pure) may be isolated such that less than 10%, particularlyless than about 1%, for example less than about 0.1% of the other isomeris present.

It will be understood by those skilled in the art that certaindiastereoisomers may be less active than others and that the activity ofan individual diastereoisomer may fall below a selected limit.

Separation of isomers may be achieved by conventional techniques knownto those skilled in the art, e.g. by fractional crystallisation,chromatography, HPLC or a combination of these techniques.

Compounds of formula (I) may exist in one of several tautomeric forms.It will be understood that the present invention encompasses alltautomers of the compounds of formula (I) whether as individualtautomers or as mixtures thereof.

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, isomers and polymorphic forms of thecompounds of formula (I) and salts thereof.

Compound Preparation

The compounds of the invention may be made by a variety of methods,including standard chemistry. Any previously defined variable willcontinue to have the previously defined meaning unless otherwiseindicated. Illustrative general synthetic methods are set out below andthen specific compounds of the invention are prepared in the workingExamples.

It will be appreciated by those skilled in the art that the (E) or (Z)description of some intermediate compounds which can exist in twogeometrical isomers, may contain the other geometric isomer as a minorcomponent.

Compounds of structural formula (I) may be prepared by a processinvolving first deprotection of a compound of structural formula (II),i.e. cleavage of the ester group, followed optionally by conversion to asalt:

where R² is a C₁-C₆ alkyl group for example a tert-butyl, ethyl ormethyl group. Alternatively R² is a chiral alkyl for example (−)-menthyl[(1R,2S,5R)-2-isopropyl-5-methylcyclohexanol].

A sixth aspect of the invention provides a compound of formula (II).

The deprotection of compound of structural formula (II) where R² ismethyl, ethyl, a chiral alkyl such as menthyl or tert-Bu may beaccomplished by acid hydrolysis using for example hydrochloric,hydrobromic, sulphuric, or trifluoroacetic acid, in an inert solvent,such as dichloromethane, 2-methyl-tetra hydrofuran, tetrahydrofuran,1,4-dioxane or cyclopentyl methyl ether or water.

Alternatively the deprotection of compound of structural formula (II)where R² is methyl, ethyl or a chiral alkyl such as menthyl may beaccomplished by base hydrolysis using for example lithium hydroxide,sodium hydroxide, potassium hydroxide in a suitable solvent, e.g. anaqueous solvent such as aqueous methanol.

After the cleavage of the ester group the resulting product may beconverted to the required salt by methods well known to those skilled inthe art.

In one embodiment the conversion of the zwitterion to the hydrochlorideslat is achieved by treatment of a solution of the zwitterion in aninert organic solvent such as acetonitrile or acetone with an aqueoushydrochloric acid solution, concentration of the resulting salt solutionand crystallisation from acetonitrile.

In one embodiment the conversion of the zwitterion to the maleate saltis achieved by treatment of an acetonitrile solution of the zwitterionwith an aqueous solution of maleic acid, heating the resulting solutionto 40° C. and allowing to cool to 5° C. for crystallisation to occur.

Compounds of structural formula (II) may be obtained from compounds ofstructural formula (III):

where R² is as defined above, by reaction with a boronic acid compoundof structural formula (IV):

Alternatively a boronate ester, such as pinacol ester may be used, whichprovides the parent boronic acid in situ. Compounds of structuralformula (IV) are commercially available e.g. from Enamine LLC, PrincetonCorporate Plaza, 7 Deer Park Drive Ste. 17-3, Monmouth Jct. NJ (USA)08852, Manchester Organics or Fluorochem. The reaction between thecompound of structural formulae (III) and (IV) may be performed in thepresence of a suitable catalyst, such as a rhodium catalyst, for examplethe dimer of rhodium (1,5-cyclooctadiene) chloride, [Rh(COD)Cl]₂ and anadditive such as a phosphine ligand, for examplebis(diphenylphosphino)-1,1′-binaphthyl (BINAP), preferably in thepresence of a base, such as aqueous potassium hydroxide, at elevatedtemperature, such as 50-90° C., and in a water-miscible solvent, such as1,4-dioxane. The reaction is preferably carried out under strictlyanaerobic conditions, where the reaction mixture is purged with an inertgas such as nitrogen, and evacuated under reduced pressure, repeatingthis process of evacuation and purging with nitrogen three times. Thisreaction produces a mixture of isomers, normally in the ratio of 1:1.The mixture of isomers produced can be separated by chromatography, HPLCor by crystallisation. An asymmetric synthesis can be achieved byinclusion of one enantiomer of the chiral ligand 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (“BINAP”) in the presence of acatalyst based on a rhodium compound. The geometry of the double bond inthe compound of structural formula (III) may be (E) or mixture of (E)and (Z) isomers, preferably pure (E) isomer.

The reaction between one enantiomer of a compound of formula (III) witha compound of formula (IV) produces two diastereoisomers inapproximately 1:1 ratio, which can be separated by crystallisation,chromatography, or by HPLC. A preferred method of separation is chiralHPLC on a chiral support, such as Chiralpak or Chiralcel columns. Theratio of the diastereoisomers formed can be increased substantially tofor example approximately 80:20 or higher, in the presence of about 10%of additives, such as(R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene [(R)-BINAP],which provides as the major isomer the biologically more activediastereoisomer.

Alternatively, various combinations of compound (III) with differentchiral R² groups, ligand, boronic acid (IV), catalyst and solventselected by those skilled in the art or by screening large numbers ofcombinations may afford a higher ratio of diastereoisomers.

The diastereoisomeric ratio may be further increased to, for example,greater than 99:1, by chiral HPLC, or by crystallisation.

Compounds of structural formula (III) may be obtained from compounds ofstructural formula (V):

by reaction with a compound of structural formula (VI)

where R² is as defined above, in the presence of an organic base such asN,N-diisopropylethylamine (“DIPEA”) and a suitable palladium-basedcatalyst, for example PdCl₂(dppf)-CH₂Cl₂ [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane, in asolvent such as dichloromethane. The compound of formula (V) can be usedas the parent compound, or be generated in situ from a salt, such as thedihydrochloride salt, in the presence of a tertiary amine base.

Compounds of structural formula (VI) may be prepared by methodsdescribed herein. By way of illustration compound of structural formula(VI), where R² is methyl, and the double bond having the (E) geometry,can be prepared by the method shown below, starting from thecommercially available methyl 4-bromocrotonate and sodium or potassiumacetate in acetonitrile at elevated temperature e.g. 50° C.:

Compounds of structural formula (V) may be prepared from compounds ofstructural formula (VII):

by catalytic hydrogenolysis for example using a palladium catalystdeposited on carbon, in an inert solvent, such as ethanol or ethylacetate.

Compounds of structural formula (VII) may be obtained from compounds ofstructural formula (VIII):

by diimide reduction, generated for example from benzenesulfonylhydrazide in the presence of a base, such as potassium carbonate, in asuitable solvent, such as DMF, and at elevated temperature, such as 130°C.

Compounds of structural formula (VIII) exist as geometrical isomers e.g.(E) or (Z)-form and may be used either as pure isomers or as mixtures.Compounds of structural formula (VIII) may be obtained starting fromknown commercially available (e.g. from Wuxi App Tec, 288 Fute ZhongRoad, Waigaoquiao Free Trade, Shanghai 200131, China) compounds ofstructural formula (IX):

which may be oxidised e.g. with sulphur trioxide in pyridine to thecorresponding aldehyde of structural formula (X):

This compound of structural formula (X) may then be reacted, which maybe without isolation of the compound of formula (X), with an ylide ofstructural formula (XI):

to thereby form the compound of formula (VIII) which exists as a mixtureof geometrical isomers (E) and (2). It will be appreciated by thoseskilled in the art that there are other methods for forming compound offormula (VIII) from the aldehyde (X). The geometrical isomers can beseparated by chromatography or used in the next step as a mixture. Thisoverall scheme for preparation of compounds of structural formula (I) issummarised below as Scheme (I):

Ylide of structural formula (XI) may be made starting from compound offormula (XII) (available from Fluorochem):

which by reaction with first hydrochloric acid followed byneutralisation with sodium bicarbonate may then be converted into analdehyde of structural formula (XIII):

which may be reduced e.g. using sodium borohydride to the correspondingalcohol of structural formula (XIV):

(See also the routes disclosed in US-A-20040092538 for preparation ofalcohols of formula (XIV).) which may then be brominated e.g. usingphosphorus tribromide to produce the corresponding bromo compound ofstructural formula (XV):

which may be converted to the triphenylphosphonium bromide (XVI) byreacting with triphenylphosphine in a solvent such as acetonitrile.

The above-mentioned ylide compound of structural formula (XI) may beobtained by reaction of compound of structural formula (XVI) with abase, such as a solution of potassium tert-butoxide in an inert solvent,such as THF. The ylide of structural formula (XI) may be isolated orpreferably formed in situ and reacted in the same vessel with analdehyde of structural formula (X) without prior isolation.

This overall scheme for preparation of ylide of structural formula (XI)is summarised below as Scheme (II):

Each of the two commercially available enantiomers of compound offormula (IX) provides one major diastereoisomer of compound of formula(I) which is more potent than the corresponding minor one.

It will be appreciated that in any of the routes described above it maybe advantageous to protect one or more functional groups. Examples ofprotecting groups and the means for their removal can be found in T. W.Greene ‘Protective Groups in Organic Synthesis’ (3rd edition, J. Wileyand Sons, 1999). Suitable amine protecting groups include acyl (e.g.acetyl), carbamate (e.g. 2′,2′,2′-trichloroethoxycarbonyl,benzyloxycarbonyl or t-butoxycarbonyl) and arylalkyl (e.g. benzyl),which may be removed by hydrolysis (e.g. using an acid such ashydrochloric acid in dioxane or trifluoroacetic acid in dichloromethane)or reductively (e.g. hydrogenolysis of a benzyl or benzyloxycarbonylgroup or reductive removal of a 2′,2′,2′-trichloroethoxycarbonyl groupusing zinc in acetic acid) as appropriate. Other suitable amineprotecting groups include trifluoroacetyl (—COCF₃) which may be removedby base catalysed hydrolysis.

It will be appreciated that in any of the routes described above, theprecise order of the synthetic steps by which the various groups andmoieties are introduced into the molecule may be varied. It will bewithin the skill of the practitioner in the art to ensure that groups ormoieties introduced at one stage of the process will not be affected bysubsequent transformations and reactions, and to select the order ofsynthetic steps accordingly.

Compounds of formulae (III), (V) to (VIII), (X), (XI), (XV) and (XVI)are also believed to be novel and therefore form a yet further aspect ofthe invention

The absolute configuration of compounds of formula (I) may be obtainedfollowing an independent enantioselective synthesis from an intermediateof known absolute configuration.

Alternatively an enantiomerically pure compound of formula (I) may beconverted into a compound whose absolute configuration is known. Ineither case comparison of spectroscopic data, optical rotation andretention times on an analytical HPLC column may be used to confirmabsolute configuration. A third option where feasible is determinationof absolute configuration through X-Ray crystallography.

Methods of Use

The compounds of formula (I) and salts thereof are believed to haveα_(v) integrin antagonist activity, particularly α_(v)β₆ receptoractivity, and thus have potential utility in the treatment of diseasesor conditions for which an α_(v)β₆ antagonist is indicated.

The present invention thus provides a compound of formula (I) or apharmaceutically acceptable salt thereof for use in therapy. Thecompound of formula (I) or pharmaceutically acceptable salt thereof canbe for use in the treatment of a disease or condition for which anα_(v)β₆ integrin antagonist is indicated.

The present invention thus provides a compound of formula (I) or apharmaceutically acceptable salt thereof for use in the treatment of adisease or condition for which an α_(v)β₆ integrin antagonist isindicated.

Also provided is the use of a compound of formula (I) or apharmaceutically acceptable salt thereof in the manufacture of amedicament for the treatment of a disease or condition for which anα_(v)β₆ integrin antagonist is indicated.

Also provided is a method of treating a disease or conditions for whichan α_(v)β₆ integrin antagonist is indicated in a subject in need thereofwhich comprises administering a therapeutically effective amount ofcompound of formula (I) or a pharmaceutically acceptable salt thereof.

Suitably the subject in need thereof is a mammal, particularly a human.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

Fibrotic diseases involve the formation of excess fibrous connectivetissue in an organ or tissue in a reparative or reactive process.α_(v)β₆ antagonists are believed to be useful in the treatment of avariety of such diseases or conditions including those dependent onα_(v)β₆ integrin function and on activation of transforming growthfactor beta via alpha v integrins. Diseases may include but are notlimited to pulmonary fibrosis (e.g. idiopathic pulmonary fibrosis,non-specific interstitial pneumonia (NSIP), usual interstitial pneumonia(UIP), Hermansky-Pudlak syndrome, progressive massive fibrosis (acomplication of coal workers' pneumoconiosis), connective tissuedisease-related pulmonary fibrosis, airway fibrosis in asthma and COPD,ARDS associated fibrosis, acute lung injury, radiation-induced fibrosis,familial pulmonary fibrosis, pulmonary hypertension); renal fibrosis(diabetic nephropathy, IgA nephropathy, lupus nephritis, focal segmentalglomerulosclerosis (FSGS), transplant nephropathy, autoimmunenephropathy, drug-induced nephropathy, hypertension-related nephropathy,nephrogenic systemic fibrosis); liver fibrosis (virally-induced fibrosis(e.g. hepatitis C or B), autoimmune hepatitis, primary biliarycirrhosis, alcoholic liver disease, non-alcoholic fatty liver diseaseincluding non-alcoholic steatohepatitis (NASH), congential hepaticfibrosis, primary sclerosing cholangitis, drug-induced hepatitis,hepatic cirrhosis); skin fibrosis (hypertrophic scars, scleroderma,keloids, dermatomyositis, eosinophilic fasciitis, Dupytrens contracture,Ehlers-Danlos syndrome, Peyronie's disease, epidermolysis bullosadystrophica, oral submucous fibrosis); ocular fibrosis (age-relatedmacular degeneration (AMD), diabetic macular oedema, dry eye, glaucoma)corneal scarring, corneal injury and corneal wound healing, preventionof filter bleb scarring post trabeculectomy surgery; cardiac fibrosis(congestive heart failure, atherosclerosis, myocardial infarction,endomyocardial fibrosis, hypertrophic cardiomyopathy (HCM)) and othermiscellaneous fibrotic conditions (mediastinal fibrosis, myelofibrosis,retroperitoneal fibrosis, Crohn's disease, neurofibromatosis, uterineleiomyomas (fibroids), chronic organ transplant rejection. There may beadditional benefits for additional inhibition of α_(v)β₁, α_(v)β₅ orα_(v)β₈ integrins

In addition, pre-cancerous lesions or cancers associated with α_(v)β₆integrins may also be treated (these may include but are not limited toendometrial, basal cell, liver, colon, cervical, oral, pancreas, breastand ovarian cancers, Kaposi's sarcoma, Giant cell tumours and cancerassociated stroma). Conditions that may derive benefit from effects onangiogenesis may also benefit (e.g. solid tumours).

The term “disease or condition for which an α_(v)β₆ antagonist isindicated”, is intended to include any or all of the above diseasestates.

In one embodiment the disease or condition for which an α_(v)β₆antagonist is indicated is idiopathic pulmonary fibrosis.

In another embodiment the disease or condition for which an α_(v)β₆antagonist is indicated is selected from corneal scarring, cornealinjury and corneal wound healing.

Compositions

While it is possible that for use in therapy, a compound of formula (I)as well as pharmaceutically acceptable salts thereof may be administeredas the raw chemical, it is common to present the active ingredient as apharmaceutical composition.

The present invention therefore provides in a further aspect apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt and one or more pharmaceuticallyacceptable carriers, diluents and/or excipients. The compounds of theformula (I) and pharmaceutically acceptable salts are as describedabove. The carrier(s), diluent(s) or excipient(s) must be acceptable inthe sense of being compatible with the other ingredients of thecomposition and not deleterious to the recipient thereof.

In accordance with another aspect of the invention there is alsoprovided a process for the preparation of a pharmaceutical compositionincluding admixing a compound of the formula (I), or a pharmaceuticallyacceptable salt thereof, with one or more pharmaceutically acceptablecarriers, diluents or excipients. The pharmaceutical composition can befor use in the treatment of any of the conditions described herein.

Further provided is a pharmaceutical composition for the treatment ofdiseases or conditions for which an α_(v)β₆ integrin antagonist isindicated comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof.

Further provided is a pharmaceutical composition comprising 0.01 to 3000mg of a compound of formula (I) or a pharmaceutical salt thereof and 0.1to 2 g of one or more pharmaceutically acceptable carriers, diluents orexcipients.

Since the compounds of formula (I) are intended for use inpharmaceutical compositions it will be readily understood that they areeach preferably provided in substantially pure form, for example, atleast 60% pure, more suitably at least 75% pure and preferably at least85% pure, especially at least 98% pure (% in a weight for weight basis).

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered more than once a day.Preferred unit dosage compositions are those containing a daily dose orsub-dose (for administration more than once a day), as herein aboverecited, or an appropriate fraction thereof, of an active ingredient.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, inhaled, intranasal, topical (including buccal,sublingual or transdermal), vagina, ocular or parenteral (includingsubcutaneous, intramuscular, intravenous or intradermal) route. Suchcompositions may be prepared by any method known in the art of pharmacy,for example by bringing into association the active ingredient with thecarrier(s) or excipient(s).

In one embodiment the pharmaceutical composition is adapted for oraladministration.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders suitable for incorporating intotablets or capsules may be prepared by reducing the compound to asuitable fine particle size (e.g. by micronisation) and mixing with asimilarly prepared pharmaceutical carrier such as an ediblecarbohydrate, as, for example, starch or mannitol. Flavouring,preservative, dispersing and colouring agent can also be present.

Capsules may be made by preparing a powder mixture, as described above,and filling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilising agent such asagaragar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, glidants,lubricants, sweetening agents, flavours, disintegrating agents andcolouring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like.

Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like.

Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum and the like. Tablets are formulated, forexample, by preparing a powder mixture, granulating or slugging, addinga lubricant and disintegrant and pressing into tablets. A powder mixtureis prepared by mixing the compound, suitably comminuted, with a diluentor base as described above, and optionally, with a binder such ascarboxymethylcellulose, an alginate, gelatin, or polyvinyl pyrrolidone,a solution retardant such as paraffin, a resorption accelerator such asa quaternary salt and/or an absorption agent such as bentonite, kaolinor dicalcium phosphate. The powder mixture can be granulated by wettingwith a binder such as syrup, starch paste, acadia mucilage or solutionsof cellulosic or polymeric materials and forcing through a screen. As analternative to granulating, the powder mixture can be run through thetablet machine and the result is imperfectly formed slugs broken intogranules. The granules can be lubricated to prevent sticking to thetablet forming dies by means of the addition of stearic acid, a stearatesalt, talc or mineral oil. The lubricated mixture is then compressedinto tablets. The compounds of the present invention can also becombined with a free flowing inert carrier and compressed into tabletsdirectly without going through the granulating or slugging steps. Aclear or opaque protective coating consisting of a sealing coat ofshellac, a coating of sugar or polymeric material and a polish coatingof wax can be provided. Dyestuffs can be added to these coatings todistinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavoured aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavour additive suchas peppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of the invention can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base. The compounds of thisinvention can be administered as topical eye drops. The compounds ofthis invention can be administered via sub-conjunctival, intracameral orintravitreal routes which would necessitate administration intervalsthat are longer than daily.

Pharmaceutical formulations adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.Formulations to be administered to the eye will have ophthalmicallycompatible pH and osmolality. One or more ophthalmically acceptable pHadjusting agents and/or buffering agents can be included in acomposition of the invention, including acids such as acetic, boric,citric, lactic, phosphoric and hydrochloric acids; bases such as sodiumhydroxide, sodium phosphate, sodium borate, sodium citrate, sodiumacetate, and sodium lactate; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases, and bufferscan be included in an amount required to maintain pH of the compositionin an ophthalmically acceptable range. One or more ophthalmicallyacceptable salts can be included in the composition in an amountsufficient to bring osmolality of the composition into an ophthalmicallyacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions.

The ocular delivery device may be designed for the controlled release ofone or more therapeutic agents with multiple defined release rates andsustained dose kinetics and permeability. Controlled release may beobtained through the design of polymeric matrices incorporatingdifferent choices and properties of biodegradable/bioerodable polymers(e.g. poly(ethylene vinyl) acetate (EVA), superhydrolyzed PVA),hydroxyalkyl cellulose (HPC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), polycaprolactone, poly(glycolic) acid, poly(lactic)acid, polyanhydride, of polymer molecular weights, polymercrystallinity, copolymer ratios, processing conditions, surface finish,geometry, excipient addition and polymeric coatings that will enhancedrug diffusion, erosion, dissolution and osmosis.

Formulations for drug delivery using ocular devices may combine one ormore active agents and adjuvants appropriate for the indicated route ofadministration. For example, the active agents may be admixed with anypharmaceutically acceptable excipient, lactose, sucrose, starch powder,cellulose esters of alkanoic acids, stearic acid, talc, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,and/or polyvinyl alcohol, tableted or encapsulated for conventionaladministration. Alternatively, the compounds may be dissolved inpolyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers. The compounds may also be mixedwith compositions of both biodegradable and non-biodegradable polymersand a carrier or diluent that has a time delay property. Representativeexamples of biodegradable compositions can include albumin, gelatin,starch, cellulose, dextrans, polysaccharides, poly (D, L-lactide), poly(D, L-lactide-co-glycolide), poly (glycolide), poly (hydroxybutyrate),poly (alkylcarbonate) and poly (orthoesters) and mixtures thereof.Representative examples of non-biodegradable polymers can include EVAcopolymers, silicone rubber and poly (methylacrylate), and mixturesthereof.

Pharmaceutical compositions for ocular delivery also include in situgellable aqueous composition. Such a composition comprises a gellingagent in a concentration effective to promote gelling upon contact withthe eye or with lacrimal fluid. Suitable gelling agents include but arenot limited to thermosetting polymers. The term “in situ gellable” asused herein includes not only liquids of low viscosity that form gelsupon contact with the eye or with lacrimal fluid, but also includes moreviscous liquids such as semi-fluid and thixotropic gels that exhibitsubstantially increased viscosity or gel stiffness upon administrationto the eye. See, for example, Ludwig (2005) Adv. Drug Deliv. Rev. 3;57:1595-639, herein incorporated by reference for purposes of itsteachings of examples of polymers for use in ocular drug delivery.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or as enemas.

Dosage forms for nasal or inhaled administration may conveniently beformulated as aerosols, solutions, suspensions, gels or dry powders.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunitdose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

Pharmaceutical compositions adapted for sub-cutaneous or intramuscularadministration include poly(lactic-co-glycolic acid) (PLGA) copolymer toform microparticles containing the active pharmaceutical ingredient toprovide sustain release.

A therapeutically effective amount of a compound of the presentinvention will depend upon a number of factors including, for example,the age and weight of the subject, the precise condition requiringtreatment and its severity, the nature of the formulation, and the routeof administration, and will ultimately be at the discretion of theattendant physician or veterinarian. In the pharmaceutical composition,each dosage unit for oral or parenteral administration preferablycontains from 0.01 to 3000 mg, more preferably 0.1 to 2000 mg, of acompound of the invention calculated as the zwitterion parent compound.

The pharmaceutically acceptable compounds of the invention can beadministered in a daily dose (for an adult patient) of, for example, anoral or parenteral dose of 0.01 mg to 3000 mg per day or 0.5 to 1000 mgper day of the compound of the formula (I) or a pharmaceuticallyacceptable salt thereof, calculated as the zwitterion. This amount maybe given in a single dose per day or more usually in a number (such astwo, three, four, five or six) of sub-doses per day such that the totaldaily dose is the same. An effective amount of a salt thereof may bedetermined as a proportion of the effective amount of the compound offormula (I) per se.

The compounds of the invention may be employed alone or in combinationwith other therapeutic agents. Combination therapies according to thepresent invention thus comprise the administration of at least onecompound of formula (I) or a pharmaceutically acceptable salt thereof,and the use of at least one other pharmaceutically active agent.Preferably, combination therapies according to the present inventioncomprise the administration of at least one compound of formula (I) or apharmaceutically acceptable salt thereof, and at least one otherpharmaceutically active agent. The compound(s) of the invention and theother pharmaceutically active agent(s) may be administered together in asingle pharmaceutical composition or separately and, when administeredseparately this may occur simultaneously or sequentially in any order.The amounts of the compound(s) of the invention and the otherpharmaceutically active agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect.

Thus in a further aspect, there is provided a combination comprising acompound of the invention and at least one other pharmaceutically activeagent.

Thus in one aspect, the compound and pharmaceutical compositionsaccording to the invention may be used in combination with or includeone or more other therapeutic agents, including therapies for allergicdisease, inflammatory disease, autoimmune disease, anti-fibrotictherapies and therapies for obstructive airway disease, therapies fordiabetic ocular diseases, and therapies for corneal scarring, cornealinjury and corneal wound healing.

Anti-allergic therapies include antigen immunotherapy (such ascomponents and fragments of bee venom, pollen, milk, peanut, CpG motifs,collagen, other components of extracellular matrix which may beadministered as oral or sublingual antigens), anti-histamines (such ascetirizine, loratidine, acrivastine, fexofenidine, chlorphenamine), andcorticosteroids (such as fluticasone propionate, fluticasone furoate,beclomethasone dipropionate, budesonide, ciclesonide, mometasonefuroate, triamcinolone, flunisolide, prednisolone, hydrocortisone).

Anti-inflammatory therapies include NSAIDs (such as aspirin, ibuprofen,naproxen), leukotriene modulators (such as montelukast, zafirlukast,pranlukast), and other anti-inflammatory therapies (such as iNOSinhibitors, tryptase inhibitors, IKK2 inhibitors, p38 inhibitors(losmapimod, dilmapimod), elastase inhibitors, beta2 agonists, DP1antagonists, DP2 antagonists, pI3K delta inhibitors, ITK inhibitors, LP(lysophosphatidic) inhibitors or FLAP (5-lipoxygenase activatingprotein) inhibitors (such as sodium3-(3-(tert-butylthio)-1-(4-(6-ethoxypyridin-3-yl)benzyl)-5-((5-methylpyridin-2-yl)methoxy)-1H-indol-2-yl)-2,2-dimethylpropanoate);adenosine a2a agonists (such as adenosine and regadenoson), chemokineantagonists (such as CCR3 antagonists or CCR4 antagonists), mediatorrelease inhibitors.

Therapies for autoimmune disease include DMARDS (such as methotrexate,leflunomide, azathioprine), biopharmaceutical therapies (such asanti-IgE, anti-TNF, anti-interleukins (such as anti-IL-1, anti-IL-6,anti-IL-12, anti-IL-17, anti-IL-18)), receptor therapies (such asetanercept and similar agents); antigen non-specific immunotherapies(such as interferon or other cytokines/chemokines, cytokine/chemokinereceptor modulators, cytokine agonists or antagonists, TLR agonists andsimilar agents).

Other anti-fibrotic therapies includes inhibitors of TGFβ synthesis(such as pirfenidone), tyrosine kinase inhibitors targeting the vascularendothelial growth factor (VEGF), platelet-derived growth factor (PDGF)and fibroblast growth factor (FGF) receptor kinases (such as Nintedanib(BIBF-1120) and imatinib mesylate (Gleevec)), endothelin receptorantagonists (such as ambrisentan or macitentan), antioxidants (such asN-acetylcysteine (NAC); broad-spectrum antibiotics (such ascotrimoxazole, tetracyclines (minocycline hydrochloride)),phosphodiesterase 5 (PDE5) inhibitors (such as sildenafil), anti-αvβxantibodies and drugs (such as anti-αvβ6 monoclonal antibodies (such asthose described in WO2003100033A2); intetumumab; cilengitide) may beused in combination.

Therapies for obstructive airway diseases include bronchodilators suchas short-acting β2-agonists, such as salbutamol), long-actingβ2-agonists (such as salmeterol, formoterol and vilanterol),short-acting muscarinic antagonists (such as ipratropium bromide),long-acting muscarinic antagonists, (such as tiotropium, umeclidinium).

In some embodiments, treatment can also involve combination of acompound of this invention with other existing modes of treatment, forexample existing agents for treatment of diabetic ocular diseases, suchas anti VEGF therapeutics e.g. Lucentis®, Avastin®, and Aflibercept® andsteroids, e.g., triamcinolone, and steroid implants containingfluocinolone acetonide.

In some embodiments, treatment can also involve combination of acompound of this invention with other existing modes of treatment, forexample existing agents for treatment of corneal scarring, cornealinjury or corneal wound healing, such as Gentel®, calf blood extract,Levofloxacin®, and Ofloxacin®.

The compounds and compositions of the present invention may be used totreat cancers alone or in combination with cancer therapies includingchemotherapy, radiotherapy, targeted agents, immunotherapy and cell orgene therapy.

It will be clear to a person skilled in the art that, where appropriate,the other therapeutic ingredient(s) may be used in the form of salts,for example as alkali metal or amine salts or as acid addition salts, orprodrugs, or as esters, for example lower alkyl esters, or as solvates,for example hydrates, to optimise the activity and/or stability and/orphysical characteristics, such as solubility, of the therapeuticingredient. It will be clear also that, where appropriate, thetherapeutic ingredients may be used in optically pure form.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention. The individual compounds of such combinationsmay be administered either sequentially or simultaneously in separate orcombined pharmaceutical compositions. Preferably, the individualcompounds will be administered simultaneously in a combinedpharmaceutical composition. Appropriate doses of known therapeuticagents will be readily appreciated by those skilled in the art.

It will be appreciated that when the compound of the present inventionis administered in combination with one or more other therapeuticallyactive agents normally administered by the inhaled, intravenous, oral,intranasal, ocular topical or other route that the resultantpharmaceutical composition may be administered by the same route.Alternatively, the individual components of the composition may beadministered by different routes.

The present inventions will now be illustrated by way of example only.

Abbreviations

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art.

-   Ac (acetyl)-   BCECF-AM (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein    acetoxymethyl ester)-   BEH (Ethylene Bridged Hybrid Technology)-   Bu (butyl)-   CBZ (carboxybenzyl)-   CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)-   Chiralcel OD-H (cellulose tris(3,5-dimethylphenylcarbamate) coated    on 5·m silica gel)-   Chiralpak AD-H (amylose tris(3,5-dimethylphenylcarbamate) coated on    5·m silica gel)-   Chiralpak ID (amylose tris(3-chlorophenylcarbamate) immobilised on    5·m silica gel)-   Chiralpak AS (amylose tris((5)-alpha-methylbenzylcarbamate) coated    on 5·m silica gel)-   CDI (carbonyl diimidazole)-   CSH (Charged Surface Hybrid Technology)-   CV (column volume)-   DCM (dichloromethane)-   DIPEA (diisopropylethylamine)-   DMF (N,N-dimethylformamide)-   DMSO (dimethylsulfoxide)-   DSC (differential scanning colorimetry)-   Et (ethyl)-   EtOH (ethanol)-   EtOAc (ethyl acetate)-   h (hour/hours)-   HCl (Hydrochloric acid)-   HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-   LCMS (liquid chromatography mass spectrometry)-   MDAP (mass directed auto-preparative HPLC)-   MDCK (Madin-Darby canine kidney)-   Me (methyl)-   MeCN (acetonitrile)-   MeOH (methanol)-   MS (mass spectrum)-   min minute/minutes-   PdCl₂(dppf)-CH₂Cl₂    [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex    with-   dichloromethane-   Ph (phenyl)-   ^(i)Pr (isopropyl)-   (R)-BINAP (R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene-   [Rh(COD)Cl]₂ ((chloro(1,5-cyclooctadiene)rhodium(I) dimer)-   RT (Retention Time)-   SPE (solid phase extraction)-   TBME (tert-butyl methyl ether)-   TEA (triethylamine)-   TFA (trifluoroacetic acid)-   TGA (thermal gravimetric analysis)-   THF (tetra hydrofuran)-   TLC (thin layer chromatography)-   UPLC (Ultra Performance Liquid Chromatography)

All references to brine refer to a saturated aqueous solution of sodiumchloride.

EXPERIMENTAL DETAILS

Analytical LCMS

Analytical LCMS was conducted on one of the following systems A, B or C.

The UV detection to all systems was an averaged signal from wavelengthof 220 nm to 350 nm and mass spectra were recorded on a massspectrometer using alternate-scan positive and negative modeelectrospray ionization.

Experimental details of LCMS systems A-D as referred to herein are asfollows:

System A

Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC BEH C₁₈ column

Flow Rate: 1 mL/min.

Temp.: 40° C.

-   -   Solvents: A: 10 mM ammonium bicarbonate in water adjusted to        pH10 with ammonia solution        -   B: Acetonitrile

Gradient: Time (min) A % B % 0 99 1 1.5 3 97 1.9 3 97 2.0 99 1

System B

Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC BEH C18 column

Flow Rate: 1 mL/min

Temp.: 40° C.

-   -   Solvents: A: 0.1% v/v solution of formic acid in water        -   B: 0.1% v/v solution of formic acid in acetonitrile

Gradient: Time (min) A % B % 0 97 3 1.5 0 100 1.9 0 100 2.0 97 3

System C

Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC CSH C18 column

Flow Rate: 1 mL/min.

Temp.: 40° C.

-   -   Solvents: A: 10 mM ammonium bicarbonate in water adjusted to        pH10 with ammonia solution        -   B: Acetonitrile

Gradient: Time (min) A % B % 0 97 3 1.5 5 95 1.9 5 95 2.0 97 3

System D

Column: 50 mm×2.1 mm ID, 1.7 μm Acquity UPLC BEH C18 column

Flow Rate: 1 mL/min

Temp.: 40° C.

-   -   Solvents: A: 0.1% v/v solution of trifluoroacetic acid in water        -   B: 0.1% v/v solution of trifluoroacetic acid in acetonitrile

Gradient: Time (min) A % B % 0 95 5 1.5 5 95 1.9 5 95 2.0 95 5

Intermediate 1 7-(Bromomethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine(Compound XV)

Phosphorus tribromide (0.565 mL, 5.99 mmol) was added dropwise to asuspension of (5,6, 7, 8-tetrahydro-1,8-naphthyridin-2-yl) methanol(Compound XIV)): see US20040092538, page 80) (820 mg, 4.99 mmol) inanhydrous acetonitrile (50 mL) at 0° C. under nitrogen. Upon addition adeep orange coloured precipitate formed, which turned to pale orange.The reaction mixture was stirred at 0° C. for 1 h by which time thereaction was complete. The mixture was concentrated in vacuo and theresidue was partitioned between ethyl acetate (250 mL) and a saturatedaqueous solution of NaHCO₃ (250 mL). The aqueous phase was furtherextracted with ethyl acetate (250 mL). The combined organic solutionswere passed through a hydrophobic frit and then concentrated in vacuo togive the title compound (1.05 g, 93%) as a fluffy creamy solid: LCMS(System C) RT=0.95 min, ES+ve m/z 227, 229 (M+H)⁺.

Intermediate 2 Triphenyl ((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl) phosphonium bromide (Compound (XVI))

A solution of 7-(bromomethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine(Compound (XV), Intermediate 1) (1.00 g, 4.40 mmol) in acetonitrile (98mL) was treated with triphenylphosphine (1.270 g, 4.84 mmol) and thesolution was stirred at room temperature under nitrogen overnight. Themixture was concentrated in vacuo to give a dark cream solid, which wasthen triturated with diethyl ether to give the title compound (2.139 g,99%) as a pale cream solid: LCMS (System C) RT=1.23 min, ES+ve m/z 409(M+H)⁺.

Intermediate 3 (E, Z) Benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl) vinyl)pyrrolidine-1-carboxylate, (Compound (VIII)

A stirred solution of (+)-benzyl 3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate (Compound (IX): available from Wuxi App Tec)(260 mg, 1.03 mmol) in DCM (3 mL) and DMSO (0.3 mL), under nitrogen, wastreated with DIPEA (0.896 mL, 5.13 mmol). After cooling to 0-5° C. (icebath) pyridine sulfur trioxide (327 mg, 2.05 mmol) was added portionwiseover ca. 5 min to oxidise the alcohol compound (IX) to the correspondingaldehyde compound (X) which was not isolated. The cooling bath wasremoved and stirring was continued for 0.5 h. Meanwhile a solution oftriphenyl ((5, 6, 7, 8-tetrahydro-1, 8-naphthyridin-2-yl) methyl)phosphonium bromide (Compound (XVI), for a preparation see Intermediate2) (553 mg, 1.13 mmol) in anhydrous DCM (10 mL), under nitrogen, wastreated dropwise with potassium tert-butoxide (1M in THF) (1.232 mL,1.232 mmol) over ca. 5 min resulting in an orange coloured solution.Stirring was continued for 10 min and then the aldehyde (formula (X))solution was added to the ylide solution in one shot and the mixture wasstirred at ambient temperature for 22 h. The reaction mixture wasdiluted with DCM (20 mL), washed with saturated aqueous sodiumbicarbonate (20 mL) and brine (20 mL), dried (Na₂SO₄) then evaporated invacuo. The dark brown residue was purified by chromatography on a 20 gsilica SPE cartridge and eluted with a gradient of 0-100% ethylacetate-cyclohexane over 30 min to obtain the title compound as twogeometrical isomers:

Isomer 1: a straw-coloured gum (123.4 mg, 31%), LCMS (System A) RT=1.28min, 95%, ES+ve m/z 382 (M+H)⁺ and

Isomer 2: a straw-coloured gum (121.5 mg, 31%), LCMS (System A) RT=1.22min, 91%, ES+ve m/z 382 (M+H)⁺

Overall yield=244.9 mg, 62.5%.

The configuration of Intermediate 3 was subsequently shown to be (R) andthe two geometrical isomers are: (R,E)-benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)vinyl)pyrrolidine-1-carboxylateand (R,Z)-benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)vinyl)pyrrolidine-1-carboxylate.

Intermediate 4 Benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidine-1-carboxylate(Compound (VII))

A solution of (E,Z)-benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)vinyl)pyrrolidine-1-carboxylate(Compound VIII, Intermediate 3) (244 mg, 0.640 mmol) (1:1, E:Z) in DMF(2 mL) was treated with benzenesulfonyl hydrazide (available from AlfaAesar) (275 mg, 1.60 mmol) and potassium carbonate (354 mg, 2.56 mmol).The reaction mixture was heated to 130° C. for 1 h, then allowed to cooland partitioned between DCM and water. The organic phase was washed withwater and dried through a hydrophobic frit. The organic solution wasevaporated in vacuo and the residual orange oil was purified bychromatography on a silica cartridge (20 g) eluting with a gradient of0-50% [(3:1 EtOAc-EtOH)-EtOAc] over 20 min. The appropriate fractionswere combined and evaporated in vacuo to give the title compound (150mg, 61%) as a pale yellow gum: LCMS (System A) RT=1.24 min, 90%, ES+vem/z 384 (M+H)⁺. The absolute configuration of Intermediate 4 wassubsequently shown to be (S) hence the compound is (S)-benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidine-1-carboxylate.The change from (R) in Intermediate 3 to (S) in Intermediate 4 is due tothe change in priority on removal of the double bond.

Intermediate 57-(2-(3-Fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine(Compound (V))

A stirred solution of benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidine-1-carboxylate(Compound (VII, Intermediate 4) (4.67 g, 12.2 mmol) in ethanol (70 mL)containing 10% palladium on carbon (0.50 g) was stirred under a hydrogenatmosphere for 7 h. LCMS showed incomplete deprotection and additional10% palladium on carbon (0.25 g) was added and the mixture was stirredunder a hydrogen atmosphere overnight. The reaction mixture existed as adark grey suspension so DCM was added to dissolve up the material untilthe mixture became black. The catalyst was removed by filtration througha pad of celite and the filtrate and washings were evaporated in vacuo.The residue was evaporated from DCM to obtain the title compound as anorange oil (3.28 g): LCMS (System A) RT=0.79 min, 90%, ES+ve m/z 250(M+H)⁺. The configuration of Intermediate 5 was subsequently establishedas (5) and the name of the compound is(S)-7-(2-(3-fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine.

Intermediate 6[7-(2-(3-Fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine,(Compound (V)) methanesulfonic acid salt

This salt of compound (V) may be prepared and crystallised as a methodof purification of compound (V) above.

2-Butanol (5 mL) was added to7-(2-(3-fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine(Compound (V)) (1.0 g, 4.0 mmol) and the mixture was heated untilcomplete dissolution was achieved. Methanesulfonic acid (0.260 mL, 4.01mmol) was added to the warm solution and the mixture was heated to 80°C. with stirring. The solution was then allowed to cool to ambienttemperature. No precipitation was evident immediately, so the solutionwas cooled further in a fridge (ca. 4° C.). After 3 days, a significantamount of solid was observed. The solid was isolated by filtration andwashed with cold 2-butanol, and dried further in vacuo to afford thetitle compound (600 mg, 43%) as a pale yellow solid: LCMS (System A)RT=0.80 min, 100%, ES+ve m/z 250 (M+H)⁺; Analytical Chiral HPLC on aChiralpak AD column (250 mm×4.6 mm) RT=8.41 min, 99.6% and RT=12.03 min,0.4%, eluting with 40% EtOH-heptane (containing 0.2% isopropylamine),flow rate 1 mL/min, detecting at 235 nm. The configuration ofIntermediate 6 was subsequently established as (5) and the name of thecompound is(S)-7-(2-(3-fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridinemethanesulfonic acid salt.

Intermediate 7 (E)-Methyl 4-acetoxybut-2-enoate (Compound (VI))

A suspension of sodium acetate (3.5 g, 42 mmol) in MeCN (30 mL) wastreated with methyl 4-bromocrotonate (Aldrich) (3.33 mL, 5 g, 28 mmol)and the mixture was heated to 50° C. for 3 d. The mixture was dilutedwith ether and then filtered. The solid was washed with ether and thecombined filtrate and washings was evaporated under reduced pressure.After evaporation the residue was partitioned between ether and water.The organic phase was washed with aqueous sodium bicarbonate, dried overMgSO₄, and evaporated under reduced pressure to give a pale orange oil.NMR indicated a mixture of product and starting material, therefore,sodium acetate (3.44 g, 42 mmol) was added to the residual oil, followedby MeCN (10 mL) and the mixture was heated to 70° C. over the weekend.The mixture was concentrated under reduced pressure and the residue waspartitioned between ether and water. The organic solution was washedwith water, brine, dried (MgSO₄) and filtered. The filtrate wasevaporated under reduced pressure to give the title compound (3.55 g,80%) as an orange oil: NMR δ (CDCl₃) 6.92 (1H, dt, J 16, 5 Hz), 6.01(1H, dt, J 16, 2 Hz), 4.72 (2H, dd, J 5, 2 Hz), 3.73 (3H, s), 2.10 (3H,s).

Intermediate 8 (E)-Methyl4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)but-2-enoate(Compound (III))

A mixture of (E)-methyl 4-acetoxybut-2-enoate (compound (VI), for apreparation see Intermediate 7) (127 mg, 0.802 mmol),7-(2-(3-fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine(compound (V), for a preparation see Intermediate 5) (200 mg, 0.802mmol) and PdCl₂(dppf)-CH₂Cl₂ adduct (65.7 mg, 0.080 mmol) in DCM (2 mL)was stirred at ambient temperature for 2 h. LCMS showed around 50%conversion and DIPEA (0.279 mL, 1.60 mmol) was added and the solutionstirred for 2 h at room temperature. LCMS showed almost completeconversion to the product. The material was loaded directly onto acolumn and purified by chromatography (20 g amino propyl cartridge)eluting with a gradient of 0-100% EtOAc in cyclohexane over 20 min. Theappropriate fractions were combined and evaporated to give the titlecompound (101.4 mg, 36%): LCMS (System A) RT=1.08 min, 95%, ES+ve m/z348 (M+H)⁺. The configuration of Intermediate 8 was established as (S)and the name as (S,E)-methyl4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)but-3-enoate.

Intermediate 9 (E, Z) (S)-Benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl) vinyl)pyrrolidine-1-carboxylate, (Compound (XXIII))

A stirred solution of (R)-(−)-benzyl3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate [(−)-compound (IX)](available from Wuxi App Tec) (4.18 g, 16.50 mmol) in dichloromethane(60 mL) and DMSO (5.86 mL, 83 mmol) was treated with DIPEA (14.41 mL, 83mmol) under nitrogen. After cooling to 0-5° C. in an ice bath, pyridinesulfur trioxide (5.40 g, 33.9 mmol) was added portion wise over ca. 5min. The solution turned a pale yellow colour and stirring was continuedfor ca. 0.5 h to give a yellow solution. The solution was washed withdilute HCl (50 mL) and dried (MgSO₄). Thentriphenyl((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)phosphoniumbromide (compound XVI, Intermediate 2) (8.06 g, 16.47 mmol) and a smallamount of DCM (ca. 5 mL) were added before the addition of cyclohexene(3.81 mL) to give a pale orange solution. Potassium tert-butoxide (19.80mL, 19.80 mmol) was added dropwise to this solution which resulted in acream coloured suspension. After 1 h the reaction mixture was dilutedwith DCM (200 mL), washed with saturated aqueous sodium bicarbonate (200mL) and brine (200 mL), dried (MgSO₄), then evaporated in vacuo. Thedark orange oil solidified overnight and was triturated with diethylether (ca. 30 mL), then filtered to give a cream solid and a yellowfiltrate. The filtrate was evaporated in vacuo to give an orange oil andthis was applied to a 330 g normal phase silica cartridge and elutedwith a cyclohexane/ethyl acetate gradient (0-100% ethyl acetate over 50min). Fractions 16-40 were evaporated in vacuo to give the titlecompound as a mixture of (E) and (Z) geometrical isomers (3.953 g, 63%)as a straw coloured gum: LCMS (System C) RT=1.28 min, 50% and 1.34 min,46% ES+ve m/z 382 (M+H)⁺.

Intermediate 10 (R)-Benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-napthyridin-2-yl)ethyl)pyrrolidine-1-carboxylate

A stirred solution of (E and Z)-(5)-benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)vinyl)pyrrolidine-1-carboxylate(Intermediate 9) (3.814 g, 10.00 mmol) in DMF (40 mL) was treated undernitrogen with potassium carbonate (5.53 g, 40.0 mmol), followed bybenzenesulfonohydrazide (4.38 g, 25.4 mmol) to give a yellow liquid. Themixture was heated at 100° C. for 1 h, then allowed to cool to ambienttemperature and filtered through celite. The filtrate was evaporated invacuo to give a cream coloured slurry. This was partitioned betweenwater (100 mL) and ethyl acetate (100 mL) and the organic layer furtherwashed with water (4×100 mL), dried (MgSO₄), and then evaporated invacuo to obtain a yellow oil (3.261 g). This was left on high vacuumline over the weekend (2.982 g). The oil was dissolved in the minimum ofDMSO (ca. 3 mL) and applied to a 120 g reverse phase cartridge andeluted with a gradient of 10-100% (acetonitrile containing 0.1% NH₃) in10 mM aqueous ammonium bicarbonate over 12 CV. Fractions 6-9 werepartially evaporated in vacuo to remove the acetonitrile. The remainingsolution was diluted with water (40 mL) and DCM (60 mL), then separated.The aqueous layer was further extracted with DCM (3×30 mL) and theorganic extracts were combined, dried (MgSO₄) and then evaporated invacuo to give the title compound (2.145 g, 56%) as a pale yellow oil:LCMS (System C): RT=1.25 min, ES+ve m/z 384 (M+H)⁺.

Intermediate 11(R)-7-(2-(3-fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine

A solution of (R)-benzyl3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidine-1-carboxylate(Intermediate 10) (2.334 g, 6.09 mmol) in ethanol (50 mL) was added to10% Palladium on carbon (250 mg, 0.235 mmol) and the mixture stirredunder a hydrogen atmosphere for 3 h at which point more palladium oncarbon (107.2 mg) was added. The reaction was stirred overnight. DCM(ca. 30 mL) was added and the mixture filtered through celite undernitrogen. The filtrate was evaporated in vacuo to give the titlecompound (1.575 g) as a yellow oil: LCMS (System C) RT=0.83 min, ES+vem/z 250 (M+H)⁺.

Intermediate 12 (R,E)-Methyl4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)but-2-enoate

(E)-methyl 4-acetoxybut-2-enoate (Intermediate 7, compound VI) (0.951 g,6.01 mmol),(R)-7-(2-(3-fluoropyrrolidin-3-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine(Intermediate 11) (1.520 g, 6.10 mmol), PdCl₂(dppf)-CH₂Cl₂ adduct (0.242g, 0.331 mmol) and potassium acetate (2.083 g, 21.22 mmol) weredissolved in DCM (25 mL) and the reaction mixture was stirred undernitrogen for 20 h to give an orange liquid (2.188 g). The reactionmixture was partitioned between DCM (50 mL) and water (50 mL) andextracted once more with DCM (50 mL). The combined organic phases werewashed with brine (50 mL) and dried over MgSO₄. The solvent was removedin vacuo and the residue was dissolved in DCM and purified on anaminopropyl cartridge (50 g) using a gradient of 0-100% ethylacetate-cyclohexane over 20 min. The appropriate fractions were combinedand evaporated in vacuo to give the title compound (1.59 g, 75%) as ayellow oil. LCMS (System C): RT=1.07 min, ES+ve m/z 348 (M+H)⁺.

Intermediate 13 Methyl4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-napthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoate

A suspension of (R,E)-methyl4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)but-2-enoate(Intermediate 12, compound (R)-III) (0.8 g, 2.303 mmol),(3-(2-methoxyethoxy)phenyl)boronic acid (available from ManchesterOrganics, Enamine or Combi-Blocks) (1.389 g, 7.09 mmol) andchloro(1,5-cyclooctadiene)rhodium(I) dimer (57 mg, 0.115 mmol) in1,4-dioxane (10 mL) was degassed. A solution of (R)-BINAP (0.173 g,0.278 mmol) and 3.8M potassium hydroxide (1.515 mL, 5.76 mmol) in1,4-dioxane (3.33 mL) was degassed. The latter solution was added to theformer solution and the mixture was stirred at 90° C. under nitrogen for1.5 h. The reaction mixture was allowed to cool and then partitionedbetween TBME (50 mL) and 2M hydrochloric acid (30 mL). The aqueous phasewas basified with saturated sodium bicarbonate solution and thenextracted using ethyl acetate (3×30 mL). The ethyl acetate extracts werewashed with brine and dried (MgSO₄). The solvent was removed in vacuoand the residue was dissolved in the minimum of DCM loaded onto anaminopropyl cartridge (50 g) and chromatographed eluting with a gradientof 0-50% ethyl acetate-cyclohexane over 20 mins. The appropriatefractions were combined and evaporated in vacuo to give the titlecompound as a mixture of diastereoisomers (0.7 g; ratio 86:14) as a paleyellow oil: LCMS (System C) RT=1.29 min, ES+ve m/z 500 (M+H)⁺.

PREPARATION OF EXAMPLES Example 1(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid and Example 2(R)-4-((S)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

(S,E)-Methyl4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)but-2-enoate(Intermediate 8) (101.4 mg, 0.292 mmol), 3.8M KOH (aq) (0.230 mL, 0.876mmol) and (3-(2-methoxyethoxy)phenyl)boronic acid (compound (IV) fromEnamine LLC) (172 mg, 0.876 mmol) were dissolved in 1,4-dioxane (2 mL)and the solution was degassed. [Rh(COD)Cl]₂ (7.20 mg, 0.015 mmol) and(R)-BINAP (21.81 mg, 0.035 mmol) were suspended in 1,4-dioxane (2 mL)and degassed. The former solution of the reactants was then added to thelatter catalyst solution under nitrogen. The reaction mixture was heatedand stirred (50° C. 2 h). The mixture was then loaded onto an SCXcartridge (10 g) (pre-conditioned with 1CV MeOH, 1CV MeCN), washed with10CV DMSO, 4CV MeCN, and eluted with 2M NH₃ in MeOH (4CV). The basicfraction was evaporated under reduced pressure. The residue was driedunder high vacuum for 12 h to give (S)-methyl4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoate(Compound (II)) (131.3 mg, 93%).

This methyl ester, Compound (II) was then dissolved in THF (2 mL) andaqueous 1M LiOH (1.459 mL, 1.459 mmol) added. The solution was stirredat room temperature for 18 h. LCMS showed complete hydrolysis to thecarboxylic acid and 2M HCl (0.876 mL, 1.751 mmol) was added and thesolution was loaded on to a SCX cartridge (10 g) (pre-conditioned with1CV MeOH, 1CV MeCN), washed with 4CV MeCN, and eluted with 2M NH₃ inMeOH (4CV). The basic fraction was evaporated under reduced pressure togive the crude product as a gum (127 mg, 90%). Analytical chiral HPLCRT=9.0 min, 88% and RT=13.8 min, 12% on a Chiralcel OJ-H column (4.6 mmid×25 cm) eluting with 60% EtOH (containing 0.2%isopropylamine)-heptane, flow rate=1.0 mL/min, detecting at 215 nm. Thediastereoisomeric mixture of compounds of Formula (I) was separated bypreparative chiral HPLC on Chiralcel OJ-H column (3 cm×25 cm) elutingwith 60% EtOH-heptane, flow rate=30 mL/min, detecting at 215 nm to givethe two individual diastereoisomers of the title compound.

Example 1 (78 mg, 55%): Analytical chiral HPLC RT=9.0 min, 98.7% on aChiralcel OJ-H column (4.6 mm id×25 cm) eluting with 60% EtOH(containing 0.2% isopropylamine)-heptane, flow rate=1.0 mL/min,detecting at 215 nm; LCMS (System D) RT=0.52 min, 100%, ES+ve m/z 486(M+H)⁺ and (System C) RT=0.81 min, 92%, ES+ve m/z 486 (M+H)⁺ ¹H NMR(CDCl₃, 600 MHz): δ 8.45 (br s, 1H), 7.21 (t, J=7.7 Hz, 1H), 7.16 (d,J=7.2 Hz, 1H), 6.86-6.73 (m, 3H), 6.31 (d, J=7.2 Hz, 1H), 4.12 (t, J=4.4Hz, 2H), 4.08 (br s, 1H), 3.75 (td, J=4.7, 0.8 Hz, 2H), 3.73-3.68 (m,1H), 3.47 (br s, 2H), 3.46 (d, J=1.1 Hz, 2H), 3.42 (br t, J=5.1 Hz, 2H),3.00-2.85 (m, 2H), 2.82-2.75 (m, 1H), 2.70-2.66 (m, 1H), 2.63-2.57 (m,1H), 2.73-2.55 (m, 3H), 2.49 (q, J=9.1 Hz, 1H), 2.45 (dd, J=11.9, 3.7Hz, 1H), 2.23-1.97 (m, 4H), 1.95-1.80 (m, 3H), [α]_(D) ²⁰+51 (c=0.72 inethanol).

The absolute configuration of the asymmetric centres of Example 1 wasdetermined and the compound was found to be(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid (see below).

Example 2 (10 mg, 7%): Analytical chiral HPLC RT=12.5 min, >99.5% on aChiralcel OJ-H column (4.6 mm id×25 cm) eluting with 60% EtOH(containing 0.2% isopropylamine)-heptane, flow rate=1.0 mL/min,detecting at 215 nm; LCMS (SystemC) RT=0.82 min, 84%, ES+ve m/z 486(M+H)⁺. [α]_(D) ²⁰−28 (c=0.50 in ethanol).

The absolute configuration of the asymmetric centres of Example 2 wasdetermined and the compound was found to be of structural formula(R)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

Example 3(R)-4-((R)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid and Example 4(S)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-napthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

To a solution of methyl4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoate(Intermediate 13) (100 mg, 0.200 mmol) in methanol (1 mL) was addedaqueous sodium hydroxide solution (2M, 0.500 mL) and the reactionmixture was stirred at room temperature for 2 h. The reaction mixturewas acidified to pH 7 using 2M hydrochloric acid. The solution wasdiluted with water (10 mL), extracted with ethyl acetate (3×10 mL), thecombined organic layers were washed with brine and dried (MgSO₄). Thesolvent was removed in vacuo giving a slightly sticky colourless glass(62 mg, 64%): LCMS (System A) RT=0.8 min, ES+ve m/z 486 (M+H)⁺.Analytical chiral HPLC RT=7.4 min, 16% and RT=11.8 min, 84% on aChiralcel OJ column (250 mm×4.6 mm), eluting with 60% EtOH-heptane,flow-rate 1 mL/min, detecting at 215 nm. The diastereoisomers wereseparated by preparative chiral HPLC on a Chiralcel OJ-H column (250mm×30 mm) eluting with 50% EtOH in heptane, flow-rate 30 mL/min, to givethe two diastereoisomers as Examples 3 and 4.

Example 3 (6 mg, 6%): LCMS (SystemC) RT=0.80 min, 94%, ES+ve m/z 486(M+H)⁺; Analytical chiral HPLC RT=7.2 min, >99.5% on a Chiralcel OJcolumn (250 mm×4.6 mm), eluting with 60% EtOH-heptane, flow-rate 1mL/min, detecting at 215 nm.(R)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid.Example 4 (33 mg, 34%): LCMS (SystemC) RT=0.80 min, 100%, ES+ve m/z 486(M+H)⁺; Analytical chiral HPLC RT=11.8 min, >99.5% on a Chiralcel OJcolumn (250 mm×4.6 mm), eluting with 60% EtOH-heptane, flow-rate 1mL/min, detecting at 215 nm.(S)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid.

Example 5(S)-4-((S)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid maleate salt

MeCN (100 μL) was added to(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid (Example 1) (112.7 mg) and heated to 60° C. To the solution, maleicacid (solid, ˜1 equivalent, 26.82 mg) was added along with seeds of(S)-4-(S)-(3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-morpholinophenyl)butanoicacid maleate salt which is described in our patent application filed onthe same day as this present application, and which is herebyincorporated by reference, and the solution was held at 60° C. for 3 h.The solution was cooled step-wise from 60° C. to 5° C. with an hour holdevery 5° C. and stirred at 5° C. for ˜16 h. The solids were isolated byvacuum filtration and air-dried for 15 min. The yield of crystallinemaleate salt was ˜41% (57.3 mg).

MeCN (300 μL) was added to(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid(Example 1) (298.54 mg) at room temperature. To the solution, maleicacid (solid, ˜1 equivalent, 71.05 mg) was added. The suspension washeated to 60° C. to obtain a clear solution. Seeds of maleate salt(obtained above) were added, but the seeds dissolved. The solution washeld at 60° C. for an hour and cooled slowly to ˜53° C. and re-seeded.The seeds dissolved but slowly. The solution was cooled slowly to 5° C.which led to a thick suspension. To the suspension, di-isopropyl ether(900 μL) was added and stirred at room temperature for two days. Thesolids were isolated by vacuum filtration, washed with di-isopropylether, air-dried for an hour and dried in a vacuum oven at 40° C.overnight. The yield of the crystalline maleate salt was (352.9 mg,95%).

Example 6(S)-4-((S)-3-Fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid citraconate salt

Anhydrous acetonitrile (0.1 mL) was added to(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid (Example 1) (505 mg, 1.04 mmol) which dissolved at ambienttemperature. Citraconic acid (28.4 mg, 0.218 mmol) (available fromSigma) was added and suspension heated to 60° C. to obtain a clearyellow solution, then cooled to ˜50° C. and held there for 5 min. Thesolution was cooled to 40° C. and then seeded with the maleate salt(Example 5) without stirring. The mixture was left in the fridgeovernight and then treated with diisopropyl ether (0.75 mL)—two layersobserved and returned to the fridge (˜3 to 4° C.) for 1 h, followed by 1h in the freezer (−22 to −20° C.) and returned to the fridge for 4 days.Precipitation/crystallisation had occurred and liquid was removed bypipette, and the solid air dried overnight to give the title compound(99 mg, 77%) as a white crystalline solid. ¹H NMR (600 MHz, D₂O)7.53-7.50 (m, 1H), 7.39-7.35 (m, 1H), 7.01-6.98 (m, 1H), 6.97-6.94 (m,2H), 6.57-6.55 (m, 1H), 5.84-5.82 (m, 1H), 4.21-4.18 (m, 2H), 3.81-3.79(m, 2H), 3.77-3.69 (m, 1H), 3.68-3.61 (m, 2H), 3.66-3.57 (m, 2H),3.52-3.42 (m, 1H), 3.44-3.41 (m, 2H), 3.41 (s, 3H), 3.44-3.40 (m, 1H),2.87-2.77 (m, 2H), 2.77-2.74 (m, 2H), 2.66-2.61 (m, 1H), 2.57-2.52 (m,1H), 2.44-2.35 (m, 1H), 2.29-2.17 (m, 1H), 2.28-2.16 (m, 2H), 2.01-1.97(m, 3H), 1.91-1.86 (m, 2H); Melting point: 103° C. (DSC)

Biological Assays

Cell Adhesion Assays

Reagents and methods utilised were as described [Ludbrook et al,Biochem. J. 2003, 369, 311 and Macdonald et al. ACS Med Chem Lett, 2014,5, 1207-1212 for α_(v)β₈ assay), with the following points ofclarification. The following cell lines were used, with ligands inbrackets: K562-α₅β₁ (Fibronectin), K562-α_(v)β₃ (LAP-b₁), K562-α_(v)β₅(Vitronectin), K562-α_(v)β₆ (LAP-b₁), K562-α_(v)β₅ (LAP-b₁). Thedivalent cation used to facilitate adhesion was 2 mM MgCl₂. Adhesion wasquantified by cell labelling with the fluorescent dye BCECF-AM (LifeTechnologies), where cell suspensions at 3×10⁶ cells/mL were incubatedwith 0.33 mL/mL of 30 mM BCECF-AM at 37° C. for 10 minutes, then 50μL/well were dispensed into the 96-well assay plate. At the assayconclusion cells that adhered were lysed using 50 μL/well of 0.5% TritonX-100 in H₂O to release fluorescence. Fluorescence intensity wasdetected using an Envision® plate reader (Perkin Elmer). For activeantagonists in the assay, data were fitted to a 4 parameter logisticequation for IC₅₀ determinations.

The affinity (pIC₅₀) for Example 1 in the cell Adhesion Assays was for:α_(v)β₆ pIC₅₀=7.9; α_(v)β₃ pIC₅₀=7.4; α_(v)β₅ pIC₅₀=7.4; α_(v)β₈pIC₅₀=7.5; α_(v)β₁ pIC₅₀=6.4.

The affinity (pIC₅₀) for Example 2 in the cell Adhesion Assays was for:α_(v)β₆ pIC₅₀=6.2; α_(v)β₃ pIC₅₀=5.9; α_(v)β₅ pIC₅₀=6.6; α_(v)β₈pIC₅₀=5.8

The affinity (pIC₅₀) for Example 3 in the cell Adhesion Assays was for:α_(v)β₆ pIC₅₀=5.4; α_(v)β₃ pIC₅₀=5.6; α_(v)β₅ pIC₅₀=5.0; α_(v)β₈pIC₅₀=5.3

The affinity (pIC₅₀) for Example 4 in the cell Adhesion Assays was for:α_(v)β₆ pIC₅₀=7.7; α_(v)β₃ pIC₅₀=6.3; α_(v)β₅ pIC₅₀=6.9; α_(v)β₈pIC₅₀=7.3.

Figures quoted are Mean pIC₅₀ values.

Permeability in MDCK Cells

The passive membrane permeability of Example 1 and Example 4 (both aszwitterion) was determined, in Madin-Darby Canine Kidney-multidrugresistance 1 (MDCKII-MDR1) cells, at pH 7.4 in the presence of thepotent P-glycoprotein inhibitor GF120918. Each compound was incubated induplicate at a concentration of 3-M on each test occasion. In this assaythe passive apparent permeability (P_(app)) of Example 1 was 71 nm/s(±23 nm/s; n=3 test occasions) and for Example 4 was 17 nm/s (n=2 testoccasions).

It was observed that although the two diastereoisomeric Examples 1 andExample 4, had similar affinity in vitro in the α_(v)β₆ cell adhesionassay (Example 1 pIC₅₀=7.9; Example 4 pIC₅₀=7.7) they had differentpermeability in MDCK cells (Example 1 P=71 nm/s and Example 4 P=17nm/s). This is expected to be reflected by Example 1 having a higheroral availability than Example 4 in vivo in pharmacokinetic studies.

Identification of the Absolute Configuration of Compounds of StructuralFormula (I)

Identification of the Absolute Configuration of the 3-fluoropyrrolidinAsymmetric Centre.

The synthesis of the target molecules (IA) commenced separately witheach enantiomer of intermediate of structural formula (IX). Theenantiomers of (IX) were purchased from Wuxi App Tec. The (+)-benzyl3-fluoro-3-(hydroxymethyl) pyrrolidine-1-carboxylate provided thediastereoisomer of (IA) with the highest affinity (Example 1 Isomer A).The absolute configuration of (+)-benzyl 3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate (IX) however was not known, and the followingexperiments outlined in scheme III were undertaken to establish itsconfiguration.

A racemic mixture of1-(tert-butoxycarbonyl)-3-fluoropyrrolidin-3-carboxylic acid (XVII)[Chemical Abstract Registry Number 1001754-59-1] (available from WuxiApp Tec) was converted to the N-α-methylbenzyl amide by reaction of theacid (XVII) with first carbonyl diimidazole (CDI), followed by(+)-(R)-α-methylbenzylamine. This provided a diastereoisomeric mixtureof amides (Scheme 3, compounds XVIII and XIX), separable bychromatography on silica gel (P. K. Mykhailiuk et. al. Convenientsynthesis of enantiopure (R)- and(S)-3-fluoro-3-aminomethylpyrrolidines, Tetrahedron 2014, 70,3011-3017). The configuration of the more polar isomer was establishedindependently by both Mykhailiuk and us by X-ray diffraction studies andshown to be (S)-tert-butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate[compound (XVIII)] (FIG. 1), and hence for the less polar isomer as(R)-tert-butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate[compound (XIX)]. Furthermore this provided reference materials forcomparison with the compound obtained by the sequence shown in SchemeIII. Although our X-ray data on the polar isomer [compound (XVIII)] wasin agreement with the X-ray crystal structure reported by Mykhailiuk et.al. the ¹H NMR spectrum differed from the spectrum we obtained. Thespectra for the two diastereoisomers [compounds (XVIII) and (XIX)] werevery similar; however, there was a small diagnostic difference for thepyrrolidine C4 proton. We observe it at 2.22 ppm. This was reported byMykhailiuk to be at 2.15 ppm. The (−)-enantiomer of compound ofstructural formula (IX) [(−)-benzyl3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate], which provided thediastereoisomer of (IA) Example 2 (Isomer 1) was hydrogenated over 10%Pd/C in ethanol to remove the CBZ protecting group, and the resultingamine (XX) protected with di-tert-butyl dicarbonate to give(−)-tert-butyl 3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate(XXI). The latter was oxidised with ruthenium trichloride and sodiumperiodate in acetonitrile-water. The resulting carboxylic acid (XXII)was then converted to the amide as before using CDI and(+)-(R)-α-methylbenzylamine. This amide was compared with the referenceamide samples (XVIII) and (XIX) and it was found to be identical by NMRspectroscopy, optical rotation and chiral HPLC to (R)-tert-butyl3-fluoro-3-(((R)-1-phenylethyl) carbamoyl) pyrrolidine-1-carboxylate(XIX). Since the (−)-enantiomer of (IX) used in this sequence is theisomer providing diastereoisomers (IA3) and (IA4) (Example 2) then the(+)-enantiomer of (IX), which provided (IA1) and (IA2) (Example 1) hasthe absolute configuration (S) at the pyrrolidine asymmetric centre.

Identification of the Absolute Configuration of the Benzylic AsymmetricCentre

The absolute configuration of the benzylic asymmetric centre of Example1 Isomer A was obtained by the degradation experiment shown in SchemeIV. Thus, Example 1 Isomer A was treated with methyl iodide in DCM atroom temperature overnight to quaternarise the pyrrolidine nitrogen andthen potassium carbonate was added, heated to 120° C. for 1 h in amicrowave reactor to give(S)-(+)-4-(3-(2-methoxyethoxy)phenyl)dihydrofuran-2(3H)-one (CompoundXXIV). The degradation product was compared with authentic(R)-(−)-4-(3-(2-methoxyethoxy)phenyl)dihydrofuran-2(3H)-one (CompoundXXV) prepared by addition of (3-(2-methoxyethoxy)phenyl)boronic acid tofuran-2(5H)-one using bis(norbornadiene)rhodium (I) tetrafluoroborate asthe catalyst and (R)-BINAP as the chiral ligand using the classicalHayashi asymmetric reaction (Hayashi, T. Tetrahedron Asymmetry, 1999,10, 4047-4056) and shown to be the enantiomer of the degradationproduct, establishing thus the configuration of Example 1 isomer A atits benzylic centre as (S).

Based on the above experiments to identify the absolute configuration ofeach asymmetric centre in compound of structural formula (I) theabsolute configuration of each Example is summarised as follows:

Example 1 is the compound of structural formula (IA2)(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid.

Example 2 is the compound of structural formula (IA1)(R)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid.

Example 3 is the compound of structural formula (IA3)(R)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid.

Example 4 is the compound of structural formula (IA4)(S)-4-((R)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl) pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy) phenyl) butanoic acid.

Experimental (S)-tert-butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate(Compound XVIII) and (R)-tert-Butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate(Compound XIX)

A solution of(±)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidine-3-carboxylic acid(compound XVII) [1001754-59-1] (available from Wuxi App Tec) (3.00 g,12.9 mmol) in THF (70 mL) was treated at room temperature with solid CDI(2.5 g, 15.4 mmol) and then the mixture was heated to 80° C. for 1.5 h.(R)-(+)-α-methylbenzylamine (available from Fluka) (1.6 g, 13.2 mmol)was added at this temperature and then the mixture was heated for afurther 1.5 h at 80° C. The mixture was diluted with ethyl acetate andwashed with dilute HCl, NaHCO₃, brine, dried (MgSO₄), filtered andallowed to evaporate slowly at room temperature. The mixture was finallyconcentrated under reduced pressure as no solid crystallised out. Theresidue was purified by chromatography on silica (2×100 g) cartridgeseluting with 0-25% EtOAc-cyclohexane over 40 min. The compound elutingfirst was obtained as a white foam (1.54 g, 36%): LCMS (System A)RT=1.17 min, ES+ve m/z 337 (M+H)⁺; ¹H NMR (500 MHz, CDCl₃) 1.43-1.49 (m,9H), 1.54 (d, J=7.0 Hz, 3H), 2.08-2.19 (m, 1H), 2.37-2.62 (m, 1H),3.43-3.56 (m, 1H), 3.61-3.93 (m, 3H), 5.14 (quin, J=7.1 Hz, 1H),6.71-6.76 (m, 1H), 7.27-7.39 (m, 5H) contains about 10% of the morepolar diastereoisomer; [α]_(D) ²⁰+61 (c=1.27 in MeOH); Analytical ChiralHPLC RT=7.58 min, 90%, and RT=9.53 min, 10% on a Chiralpak AD column(250 mm×4.6 mm), eluting with 10% EtOH-heptane, flow rate=1 mL/min,detecting at 215 nm. A 50 mg portion of this sample was further purifiedon a silica cartridge (20 g) eluting with 0-25% EtOAc-cyclohexane over20 min. The appropriate fraction was evaporated under reduced pressureto give an analytically pure sample (30 mg) of (R)-tert-Butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate(compound XIX) LCMS (SystemC) RT=1.16 min, ES+ve m/z 337 (M+H)⁺ and 354(M+NH₄)⁺ and ES−ve m/z 335 (M−H)⁻; [α]_(D) ²⁰+63 (c=0.933 in MeOH).

The second compound eluting from the column (more polar diastereoisomer)(1.2 g, 28%) was crystallised from ether to give white crystals of(S)-tert-butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate(compound XVIII): mp=113-115° C.; LCMS (SystemC) RT=1.16 min, ES+ve m/z337 (M+H)⁺; ¹H NMR (500 MHz, CDCl₃) 1.43-1.48 (m, 9H), 1.54 (d, J=7.0Hz, 3H), 2.14-2.26 (m, 1H), 2.44-2.70 (m, 1H), 3.46-3.55 (m, 1H),3.56-3.87 (m, 3H), 5.14 (quin, J=7.1 Hz, 1H), 6.73 (br s, 1H), 7.27-7.40(m, 5H); [α]_(D) ²⁰+73 (c=0.876 in MeOH); Analytical Chiral HPLC RT=9.50min, 100% on a Chiralpak AD column (250 mm×4.6 mm) eluting with 10%EtOH-heptane, flow rate=1 mL/min, detecting at 215 nm. The absoluteconfiguration of this diastereoisomer was established from an X-raydiffraction study.

(−)-(R)-(3-Fluoropyrrolidin-3-yl) methanol (Compound XX)

A solution of (−)-N-CBZ-3-fluoro-3-(hydroxymethyl) pyrrolidine,(−)-isomer of compound (IX), (available from Wuxi App Tec) (4.0 g, 15.8mmol) was hydrogenated over 10% Pd/C (400 mg) in ethanol (150 mL)overnight. The catalyst was removed by filtration through celite andwashed with ethanol. The filtrate and washings were evaporated underreduced pressure to give the title compound (2.0 g, 106%, contains someethanol by NMR) as a yellow oil, which solidified into a waxy solid:LCMS (SystemC) RT=0.22 min, ES+ve m/z 120 (M+H)⁺ and ES−ve m/z 118(M−H)⁻. The product was further dried in a blow-down unit under nitrogenat 40° C. ¹H NMR (500 MHz, CDCl₃) 3.82 (dd, J=18.7, 12.5 Hz, 1H), 3.73(dd, J=22.0, 12.2 Hz, 1H), 3.22-3.15 (m, 1H), 3.23-3.14 (m, 1H),2.99-2.92 (m, 1H), 2.91 (dd, J=29.1, 13.2 Hz, 1H), 2.66 (br s, 2H),2.10-1.98 (m, 1H), 1.94-1.81 (m, 1H); [α]_(D) ²⁰=−4 (c=1.19 in EtOH).

(−)-(R)-tert-Butyl 3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate(Compound XXI)

A solution of (R)-(3-fluoropyrrolidin-3-yl)methanol (compound XX) (1.88g, 15.8 mmol) in DCM (15 mL) and diisopropylethylamine (4.13 mL, 23.7mmol) was treated with di-tert-butyl dicarbonate (3.79 g, 17 mmol) andthe mixture was stirred at 20° C. for 3 h. The mixture was partitionedbetween 2M HCl and DCM and separated in a phase separator cartridge. Theorganic layer was concentrated under reduced pressure and the residuewas purified by chromatography on a silica cartridge (70 g) eluting witha gradient of 0-50% EtOAc-cyclohexane over 40 min. The fractions werechecked by TLC on silica (50% EtOAc-cyclohexane) and stained with KMnO₄solution. Appropriate fractions were combined and evaporated underreduced pressure to give the title compound (2.73 g, 79%) as acolourless oil: LCMS (SystemC) RT=0.79 min, ES+ve m/z 220 (M+H)⁺ and 439(2M+H)⁺; ¹H NMR (400 MHz, DMSO-d₆) δ 1.42 (s, 9H), 1.96-2.14 (m, 2H),3.32-3.41 (m, 2H), 3.42-3.50 (m, 2H), 3.54-3.61 (m, 1H), 3.62-3.69 (m,H), 4.90 (t, J=5.8 Hz, 1H); [α]_(D) ²⁰=−28 (c=3.51 in CHCl₃).

(R)-tert-Butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate(Compound XIX)

A solution of (−)-tert-butyl3-fluoro-3-(hydroxymethyl)pyrrolidine-1-carboxylate (compound XXI) (200mg, 0.9 mmol) in MeCN (1 mL) and water (1 mL) was treated with RuCl₃(9.5 mg, 0.05 mmol) and sodium periodate (976 mg, 4.5 mmol) and themixture was stirred at 20° C. for 16 h. The mixture was acidified with1M HCl (5 mL) and partitioned in DCM. The aqueous phase was re-extractedtwice with DCM and the phases separated in a phase-separation cartridge.The organic solution was evaporated in a blow-down unit to give(R)-1-(tert-butoxycarbonyl)-3-fluoropyrrolidine-3-carboxylic acid(compound XXII) (125 mg, 59%): MS ES−ve m/z 232 (M−H)⁻. The acid (125mg, 0.54 mmol) was dissolved in ethyl acetate (10 mL) and treated withCDI (360 mg, 2.2 mmol) and the mixture was stirred at room temperaturefor 1 h and then heated at 50° C. for 0.5 h. The mixture wasconcentrated in a blow-down unit, the residue was dissolved in THF (6mL) and treated with (R)-(+)-α-methylbenzylamine (200 mg, 1.9 mmol) andstirred at 20° C. for 1.5 h. The mixture was diluted with ethyl acetateand washed with 2M HCl solution twice, followed by brine. The organicsolution was dried (MgSO₄) and evaporated under reduced pressure to givea grey solid (290 mg). The residue was dissolved in MeOH-DMSO (1:1; 3mL) and purified by MDAP on a XSELECT CSH C18 column (150 mm×30 mm i.d.5 μm packing diameter) at ambient temperature, eluting with a gradientof 30-85% (10 mM ammonium bicarbonate in water adjusted to pH 10 withaq. ammonia solution-acetonitrile) running for 30 min, detecting at 254nm and collecting the peak with RT=17.4 min, ES+ve m/z 337 (M+H)⁺. Thefraction was concentrated in a blow-down unit at 45° C. under nitrogenand the residual suspension was extracted with EtOAc. The organicsolution was washed with 2M HCl twice and then with brine, dried (MgSO₄)and evaporated under reduced pressure to give a yellow gum (35 mg). Thegum was re-purified by MDAP on a XBridge C18 column (100 mm×19 mm i.d. 5μm packing diameter) at ambient temperature eluting with a gradient of(10 mM ammonium bicarbonate in water adjusted to pH 10 with aq. ammoniasolution-acetonitrile) running for 25 min, detecting at 254 nm)collecting the first fraction (RT=10 min). The solvent was removed in ablow-down unit under nitrogen at 45° C. to give the title compound (16mg, 5%) as a colourless gum: LCMS (System C) RT=1.16 min, ES+ve m/z 337(M+H)⁺, 354 (M+NH₄)⁻; Analytical Chiral HPLC RT=7.58 min, 97.7% on aChiralpak AD column (250 mm×4.6 mm) eluting with 10% EtOH-heptane, flowrate=1 mL/min, detecting at 215 nm; [α]_(D) ²⁰+63 (c=1.15 in MeOH). The¹H NMR spectrum (500 MHz, CDCl₃) as well as the optical rotation and thechiral HPLC RT all match those of (R)-tert-butyl3-fluoro-3-(((R)-1-phenylethyl)carbamoyl)pyrrolidine-1-carboxylate(compound XIX).

Determination of the Absolute Configuration of the Benzylic AsymmetricCentre of Example 1 by Degradation to(S)-(+)-4-(3-(2-methoxyethoxy)phenyl)dihydrofuran-2(3H)-one (CompoundXXIV)

A solution of4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid (Example 1 (200 mg, 0.412 mmol) in DCM (20 mL) was treated withiodomethane (0.400 mL, 6.40 mmol) at room temperature and stirred for 18h. The reaction mixture was concentrated in vacuo to remove the excessiodomethane, the residual solid was re-dissolved in DCM (10 mL) and thenpotassium carbonate (250 mg, 1.809 mmol) was added. The reaction mixturewas heated in a microwave reactor to 120° C. for 1 h. The solution wasfiltered and concentrated in vacuo and the residual oil was purified bychromatography on a silica column (10 g) eluting with a gradient of0-100% TBME in cyclohexane, detecting at 220 nm. The relevant fractionswere concentrated in vacuo affording(S)-(+)-4-(3-(2-methoxyethoxy)phenyl)dihydrofuran-2(3H)-one (compoundXXIV) (80 mg, 82%) as a colourless oil: LCMS (System B) RT=0.80 min,100%, ES+ve m/z 237 (M+H)⁻; ¹H NMR (400 MHz, CDCl₃) 7.34-7.26 (m, 1H),6.92-6.72 (m, 3H), 4.67 (dd, J=9.0, 7.9 Hz, 1H), 4.28 (dd, J=9.1, 8.1Hz, 1H), 4.20-4.10 (m, 2H), 3.84-3.72 (m, 3H), 3.48 (s, 3H), 2.93 (dd,J=17.5, 8.7 Hz, 1H), 2.68 (dd, J=17.5, 9.0 Hz, 1H); [α]_(D) ²²=+42(c=1.06 in CHCl₃); Chiral HPLC RT=9.72 min, 100% on a ChiralpakAD column(250 mm×4.6 mm) eluting with 40% EtOH in heptane, flow-rate=1 mL/min,detecting at 215 nm.

Synthesis of Authentic(R)-(−)-4-(3-(2-Methoxyethoxy)phenyl)dihydrofuran-2(3H)-one (CompoundXXV) for Comparison with Compound (XXIV)

To a solution of bis(norbornadiene)rhodium (I) tetrafluoroborate(available from Aldrich) (37.4 mg, 0.100 mmol), (R)-BINAP (125 mg, 0.200mmol) and (3-(2-methoxyethoxy)phenyl)boronic acid (available fromEnamine) (980 mg, 5.00 mmol) in 1,4-dioxane (10 mL) was addedfuran-2(5H)-one (available from Alfa Aesar) (0.142 mL, 2.0 mmol) andaqueous KOH (3.8 M, 1.053 mL, 4.00 mmol). The resulting solution washeated to 100° C. for 1 h in a microwave reactor. The reaction mixturewas allowed to cool and partitioned between water (20 mL) and DCM (20mL). The layers were separated and the organic layer was passed througha hydrophobic frit and concentrated in vacuo. The residual oil waspurified by chromatography on a KPNH column (50 g) eluting with agradient of 0-100% TBME in cyclohexane over 45 min, detecting at 220 nm.The relevant fractions were concentrated in vacuo to give the titlecompound (101 mg, 21%) as a colourless oil: LCMS (System B) RT=0.80 min,100%, ES+ve m/z 237 (M+H)⁻; [α]_(D) ²³=−37 (c=1.10 in CHCl₃); ChiralHPLC RT=11.82 min, 94% and RT=9.67 min, 6% on a Chiralpak AD column (250mm×4.6 mm) eluting with 40% EtOH in heptane, flow rate=1 mL/min,detecting at 215 nm, indicating that the title compound is theenantiomer of compound (XXIV).

Hence: Example 1 is(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

And Example 2 is(R)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid.

The invention claimed is:
 1. A method of treating a fibrotic disease ordisorder in a human in need thereof comprising administering to thehuman a therapeutically effective amount of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 2. A method of treating afibrotic disease or disorder in a human in need thereof comprisingadministering to the human a therapeutically effective amount of(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 3. A combinationcomprising of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof, and a therapeuticallyactive agent.
 4. A combination comprising of(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof, and a therapeuticallyactive agent.
 5. The combination according to claim 3, wherein thetherapeutically active agent is an inhibitor of TGFβ synthesis.
 6. Thecombination according to claim 4, wherein the therapeutically activeagent is an inhibitor of TGFβ synthesis.
 7. The combination according toclaim 3, wherein the therapeutically active agent is pirfenidone.
 8. Thecombination according to claim 4, wherein the therapeutically activeagent is pirfenidone.
 9. The combination according to claim 3, whereinthe therapeutically active agent is nintedanib.
 10. The combinationaccording to claim 4, wherein the therapeutically active agent isnintedanib.
 11. The combination according to claim 3, wherein thetherapeutically active agent is an anti αvβ6 receptor antibody orantigen binding fragment thereof.
 12. The combination according to claim4, wherein the therapeutically active agent is an anti αvβ6 receptorantibody or antigen binding fragment thereof.
 13. A method of treating afibrotic disease or disorder in a human in need thereof comprisingadministering a therapeutically effective amount to the human of acombination comprising4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof, and a therapeuticallyactive agent.
 14. A method of treating a fibrotic disease or disorder ina human in need thereof comprising administering a therapeuticallyeffective amount to the human of a combination comprising(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof, and a therapeuticallyactive agent.
 15. The method according to claim 13, wherein thetherapeutically active agent is an inhibitor of TGFβ synthesis.
 16. Themethod according to claim 14, wherein the therapeutically active agentis an inhibitor of TGFβ synthesis.
 17. The method according to claim 13,wherein the therapeutically active agent is an anti αvβ6 receptorantibody or antigen binding fragment thereof.
 18. The method accordingto claim 14, wherein the therapeutically active agent is an anti αvβ6receptor antibody or antigen binding fragment thereof.
 19. A method oftreating idiopathic pulmonary fibrosis in a human comprisingadministering to the human in need thereof a therapeutically effectiveamount of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 20. A method of treatingidiopathic pulmonary fibrosis in a human comprising administering to thehuman in in need thereof a therapeutically effective amount of(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 21. A method of treatingpulmonary fibrosis in a human comprising administering to the human inneed thereof a therapeutically effective amount of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 22. The method oftreating according to claim 21, wherein the pulmonary fibrosis isnon-specific interstitial pneumonia or usual interstitial pneumonia. 23.A method of treating pulmonary fibrosis in a human comprisingadministering to the human in need thereof a therapeutically effectiveamount of(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 24. The method accordingto claim 23, wherein the pulmonary fibrosis is non-specific interstitialpneumonia or usual interstitial pneumonia.
 25. A method of treatinghepatic fibrosis in a human comprising administering to the human inneed thereof a therapeutically effective amount of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 26. The method accordingto claim 25, wherein the hepatic fibrosis is viral induced fibrosis,hepatitis C, primary biliary cirrhosis, alcoholic liver disease,non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis. 27.A method of treating hepatic fibrosis in a human comprisingadministering to the human in in need thereof a therapeuticallyeffective amount of(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 28. The method accordingto claim 27, wherein the hepatic fibrosis is viral induced fibrosis,hepatitis C, primary biliary cirrhosis, alcoholic liver disease,non-alcoholic fatty liver disease, or non-alcoholic steatohepatitis. 29.A method of treating renal fibrosis in a human comprising administeringto the human in need thereof a therapeutically effective amount of4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 30. The method accordingto claim 29, wherein the renal fibrosis is diabetic nephropathy, IgAnephropathy, or focal segmental glomerulosclerosis.
 31. A method oftreating renal fibrosis in a human comprising administering to the humanin in need thereof a therapeutically effective amount of(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof.
 32. The method accordingto claim 31, wherein the renal fibrosis is diabetic nephropathy, IgAnephropathy, or focal segmental glomerulosclerosis.
 33. A method oftreating idiopathic pulmonary fibrosis in a human in need thereofcomprising administering a therapeutically effective amount to the humanof a combination comprising4-(3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof, and a therapeuticallyactive agent.
 34. A method of treating idiopathic pulmonary fibrosis ina human in need thereof comprising administering a therapeuticallyeffective amount to the human of a combination comprising(S)-4-((S)-3-fluoro-3-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)pyrrolidin-1-yl)-3-(3-(2-methoxyethoxy)phenyl)butanoicacid

or a pharmaceutically acceptable salt thereof, and a therapeuticallyactive agent.
 35. The method according to claim 33, wherein thetherapeutically active agent is pirfenidone.
 36. The method according toclaim 34, wherein the therapeutically active agent is pirfenidone. 37.The method according to claim 33, wherein the therapeutically activeagent is nintedanib.
 38. The method according to claim 34, wherein thetherapeutically active agent is nintedanib.